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

The surface chemistry of nanomaterials, particularly the density of functional groups, governs their behavior in applications such as bioanalysis, bioimaging, and environmental impact studies. Here, we report a precise method to quantify carboxyl groups per nanoparticle by combining anisotropically collapsing agarose gels for nanoparticle immobilization with fluorescence microscopy and acid-base titration. We applied this approach to photon-upconversion nanoparticles (UCNPs) coated with poly(acrylic acid) (PAA) and fluorescence-labeled polystyrene nanoparticles (PNs), which serve as models for bioimaging and environmental pollutants, respectively. UCNPs exhibited 152 ± 14 thousand carboxyl groups per particle (∼11 groups/nm2), while PNs were characterized with 38 ± 3.6 thousand groups (∼1.7 groups/nm2). The limit of detection was 6.4 and 1.9 thousand carboxyl groups per nanoparticle, and the limit of quantification was determined at 21 and 6.2 thousand carboxyl groups per nanoparticle for UCNP-PAAs and PNs, respectively. High intrinsic luminescence enabled direct imaging of UCNPs, while PNs required fluorescence staining with Nile Red to overcome low signal-to-noise ratios. The study also discussed the critical influence of nanoparticle concentration and titration conditions on the assay performance. This method advances the precise characterization of surface chemistry, offering insights into nanoparticle structure that extend beyond the resolution of electron microscopy. Our findings establish a robust platform for investigating the interplay of surface chemistry with nanoparticle function and fate in technological and environmental contexts, with broad applicability across nanomaterials.

• MeSH
• Acrylic Resins chemistry   MeSH
• Deep Learning * MeSH
• Microscopy, Fluorescence MeSH
• Gels chemistry   MeSH
• Microplastics * chemistry   MeSH
• Nanoparticles * chemistry   MeSH
• Polystyrenes chemistry   MeSH
• Sepharose chemistry   MeSH
• Particle Size MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acrylic Resins MeSH
• carbopol 940 MeSH Browser
• Gels MeSH
• Microplastics * MeSH
• Polystyrenes MeSH
• Sepharose MeSH

We present a simplistic and absolute method for estimating the number concentration of nanoparticles. Macroscopic volumes of a nanoparticle dispersion (several μL) are dropped on a glass surface and the solvent is evaporated. The optical microscope scans the entire surface of the dried droplet (several mm2), micrographs are stitched together (several tens), and all nanoparticles are counted (several thousand per droplet) by using an artificial neural network. We call this method evaporated volume analysis (EVA) because nanoparticles are counted after droplet volume evaporation. As a model, the concentration of ∼60 nm Tm3+-doped photon-upconversion nanoparticles coated in carboxylated silica shells is estimated with a combined relative standard uncertainty of 2.7%. Two reference methods provided comparable concentration values. A wider applicability is tested by imaging ∼80 nm Nile red-doped polystyrene and ∼90 nm silver nanoparticles. Theoretical limits of EVA such as the limit of detection, limit of quantification, and optimal working range are discussed.

• Publication type
• Journal Article MeSH

The anti-Stokes emission of photon upconversion nanoparticles (UCNPs) facilitates their use as labels for ultrasensitive detection in biological samples as infrared excitation does not induce autofluorescence at visible wavelengths. The detection of extremely low-abundance analytes, however, remains challenging as it is impossible to completely avoid nonspecific binding of label conjugates. To overcome this limitation, we developed a novel hybridization complex transfer technique using UCNP labels to detect short nucleic acids directly without target amplification. The assay involves capturing the target-label complexes on an initial solid phase, then using releasing oligonucleotides to specifically elute only the target-UCNP complexes and recapturing them on another solid phase. The nonspecifically adsorbed labels remain on the first solid phase, enabling background-free, ultrasensitive detection. When magnetic microparticles were used as the first solid phase in a sample volume of 120 μL, the assay achieved a limit of detection (LOD) of 310 aM, a 27-fold improvement over the reference assay without transfer. Moreover, the additional target-specific steps introduced in the complex transfer procedure improved the sequence specificity of the complex transfer assay compared with the reference assay. The suitability for clinical analysis was confirmed using spiked plasma samples, resulting in an LOD of 190 aM. By increasing the sample volume to 600 μL and using magnetic preconcentration, the LOD was improved to 46 aM. These results highlight the importance of background elimination in achieving ultralow LODs for the analysis of low-abundance biomarkers.

• MeSH
• Nucleic Acid Hybridization MeSH
• Humans MeSH
• Limit of Detection MeSH
• Luminescence MeSH
• Luminescent Measurements * methods   MeSH
• Nanoparticles * chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Photon-upconversion nanoparticles (UCNP) have already been established as labels for affinity assays in analog and digital formats. Here, advanced, or smart, systems based on UCNPs coated with active shells, fluorescent dyes, and metal and semiconductor nanoparticles participating in energy transfer reactions are reviewed. In addition, switching elements can be embedded in such assemblies and provide temporal and spatial control of action, which is important for intracellular imaging and monitoring activities. Demonstration and critical comments on representative approaches demonstrating the progress in the use of such UCNPs in bioanalytical assays, imaging, and monitoring of target molecules in cells are reported, including particular examples in the field of cancer theranostics.

• Keywords
• Bioconjugates, Fluorescent dyes, Intracellular biosensor, Nanosensor, Resonance energy transfer,
• MeSH
• Fluorescent Dyes chemistry   MeSH
• Photons * MeSH
• Humans MeSH
• Neoplasms diagnostic imaging   diagnosis   MeSH
• Nanoparticles * chemistry   MeSH
• Optical Imaging MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Fluorescent Dyes 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
• Biomarkers * blood   urine   analysis   MeSH
• Immunoassay methods   MeSH
• Quantum Dots chemistry   MeSH
• Humans MeSH
• Serum Albumin, Human analysis   urine   MeSH
• Limit of Detection * MeSH
• Nanoparticles * chemistry   MeSH
• Prostate-Specific Antigen * blood   analysis   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers * MeSH
• Serum Albumin, Human MeSH
• Prostate-Specific Antigen * 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.

• Keywords
• Covid-19 diagnosis, NIR to UV upconverting nanoparticles, Reactive oxygen species, Singlet oxygen,
• MeSH
• COVID-19 * diagnosis   MeSH
• Immunoassay MeSH
• Humans MeSH
• Nanoparticles * MeSH
• SARS-CoV-2 MeSH
• Singlet Oxygen MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Singlet Oxygen MeSH

Massively parallel spectroscopy (MPS) of many single nanoparticles in an aqueous dispersion is reported. As a model system, bioconjugated photon-upconversion nanoparticles (UCNPs) with a near-infrared excitation are prepared. The UCNPs are doped either with Tm3+ (emission 450 and 802 nm) or Er3+ (emission 554 and 660 nm). These UCNPs are conjugated to biotinylated bovine serum albumin (Tm3+-doped) or streptavidin (Er3+-doped). MPS is correlated with an ensemble spectra measurement, and the limit of detection (1.6 fmol L-1) and the linearity range (4.8 fmol L-1 to 40 pmol L-1) for bioconjugated UCNPs are estimated. MPS is used for observing the bioaffinity clustering of bioconjugated UCNPs. This observation is correlated with a native electrophoresis and bioaffinity assay on a microtiter plate. A competitive MPS bioaffinity assay for biotin is developed and characterized with a limit of detection of 6.6 nmol L-1. MPS from complex biological matrices (cell cultivation medium) is performed without increasing background. The compatibility with polydimethylsiloxane microfluidics is proven by recording MPS from a 30 μm deep microfluidic channel.

• MeSH
• Nanoparticles * chemistry   MeSH
• Spectrum Analysis MeSH
• Streptavidin MeSH
• Artificial Intelligence * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Streptavidin MeSH

The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout.

• MeSH
• COVID-19 * diagnosis   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Immunosorbents * MeSH
• Humans MeSH
• Nucleocapsid Proteins MeSH
• Antibodies, Viral MeSH
• SARS-CoV-2 MeSH
• Sensitivity and Specificity MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Immunosorbents * MeSH
• Nucleocapsid Proteins MeSH
• Antibodies, Viral MeSH

A nanocomposite consisting of a cubic EuSe semiconductor material grown on a hexagonal upconversion nanoparticle has overcome the crystal lattice mismatch that typically prevents the epitaxial growth of such heterogeneous nanocrystals. Eu3+ at the interface layer shows its characteristic red emission band both under UV excitation light due to energy transfer from the semiconductor and under NIR excitation light due to energy transfer after photon-upconversion. Data storage and security applications are suggested for this new nanocomposite.

• Publication type
• Journal Article MeSH