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

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.

• Keywords
• antifouling biointerface, biomarker, digital readout of assay, rolling circle amplification, single molecule detection, surface plasmon resonance, surface plasmon-enhanced fluorescence,
• MeSH
• DNA, Single-Stranded * MeSH
• Humans MeSH
• Surface Plasmon Resonance * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Single-Stranded * 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

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

The detection of cancer biomarkers in histological samples and blood is of paramount importance for clinical diagnosis. Current methods are limited in terms of sensitivity, hindering early detection of disease. We have overcome the shortcomings of currently available staining and fluorescence labeling methods by taking an integrative approach to establish photon-upconversion nanoparticles (UCNP) as a powerful platform for cancer detection. These nanoparticles are readily synthesized in different sizes to yield efficient and tunable short-wavelength light emission under near-infrared excitation, which eliminates optical background interference of the specimen. Here we present a protocol for the synthesis of UCNPs by high-temperature co-precipitation or seed-mediated growth by thermal decomposition, surface modification by silica or poly(ethylene glycol) that renders the particles resistant to nonspecific binding, and the conjugation of streptavidin or antibodies for biological detection. To detect blood-based biomarkers, we present an upconversion-linked immunosorbent assay for the analog and digital detection of the cancer marker prostate-specific antigen. When applied to immunocytochemistry analysis, UCNPs enable the detection of the breast cancer marker human epidermal growth factor receptor 2 with a signal-to-background ratio 50-fold higher than conventional fluorescent labels. UCNP synthesis takes 4.5 d, the preparation of the antibody-silica-UCNP conjugate takes 3 d, the streptavidin-poly(ethylene glycol)-UCNP conjugate takes 2-3 weeks, upconversion-linked immunosorbent assay takes 2-4 d and immunocytochemistry takes 8-10 h. The procedures can be performed after standard laboratory training in nanomaterials research.

• MeSH
• Immunosorbents MeSH
• Humans MeSH
• Biomarkers, Tumor MeSH
• Neoplasms * diagnosis   MeSH
• Nanoparticles * chemistry   MeSH
• Silicon Dioxide chemistry   MeSH
• Polyethylene Glycols chemistry   MeSH
• Streptavidin MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Immunosorbents MeSH
• Biomarkers, Tumor MeSH
• Silicon Dioxide MeSH
• Polyethylene Glycols MeSH
• Streptavidin MeSH

Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL-1 in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals.

• Keywords
• anti-Stokes emission, cardiac arrest, lanthanide-doped nanomaterials, single molecule immunoassay, troponin,
• MeSH
• Photons MeSH
• Immunoassay MeSH
• Humans MeSH
• Nanoparticles * MeSH
• Troponin MeSH
• Particle Size MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Troponin 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.

• Keywords
• Immunocytochemistry, Immunohistochemistry, Laser-induced breakdown spectroscopy, Photon-upconversion nanoparticles, Tag-LIBS,
• MeSH
• Fluorides chemistry   radiation effects   MeSH
• Antibodies, Immobilized immunology   MeSH
• Immunohistochemistry methods   MeSH
• Humans MeSH
• Biomarkers, Tumor analysis   immunology   MeSH
• Cell Line, Tumor MeSH
• Nanoparticles chemistry   radiation effects   MeSH
• Receptor, ErbB-2 analysis   immunology   MeSH
• Spectrum Analysis methods   MeSH
• Feasibility Studies MeSH
• Light MeSH
• Thulium chemistry   radiation effects   MeSH
• Yttrium chemistry   radiation effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ERBB2 protein, human MeSH Browser
• Fluorides MeSH
• Antibodies, Immobilized MeSH
• Biomarkers, Tumor MeSH
• Receptor, ErbB-2 MeSH
• sodium yttriumtetrafluoride MeSH Browser
• Thulium MeSH
• Yttrium MeSH

The ability to detect low concentrations of analytes and in particular low-abundance biomarkers is of fundamental importance, e.g., for early-stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single-molecule bioaffinity assays. While many review articles have highlighted the potentials of single-molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real-world applications as one should expect. This Review provides a theoretical background on single-molecule-or better digital-assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single-molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials.

• Keywords
• digital assays, immunoassays, optical detection, signal background, single-molecule detection,
• MeSH
• Biomarkers analysis   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Fluorescent Dyes chemistry   MeSH
• Limit of Detection MeSH
• Nanostructures chemistry   MeSH
• Nucleic Acids analysis   MeSH
• Polymerase Chain Reaction methods   MeSH
• Signal-To-Noise Ratio MeSH
• Proteins analysis   MeSH
• Binding Sites MeSH
• Viruses isolation & purification   MeSH
• Single Molecule Imaging methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Biomarkers MeSH
• Fluorescent Dyes MeSH
• Nucleic Acids MeSH
• Proteins 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.

• Keywords
• Collinear plasma collection, Gold nanoparticles, Laser ablation, Microtiter plate, Sandwich immunoassay, Silver nanoparticles, Streptavidin, Tag-LIBS,
• MeSH
• Biomarkers blood   MeSH
• Immunoassay methods   MeSH
• Metal Nanoparticles chemistry   MeSH
• Lasers * MeSH
• Humans MeSH
• Serum Albumin, Human analysis   MeSH
• Surface Properties MeSH
• Silver chemistry   MeSH
• Particle Size MeSH
• Gold chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers MeSH
• Serum Albumin, Human MeSH
• Silver MeSH
• Gold MeSH