Upconversion Nanoparticle-Based Dot-Blot Immunoassay for Quantitative Biomarker Detection.

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.

Digital and Analog Detection of SARS-CoV-2 Nucleocapsid Protein via an Upconversion-Linked Immunosorbent Assay.

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.

Otto S. Wolfbeis (1947-2023).

Otto Wolfbeis, Professor emeritus of Analytical Chemistry at the University of Regensburg, passed away on June 1, 2023. Along with his seminal work on optical sensors and fluorescent (nano)materials, he will be remembered as an outstanding researcher who inspired many talents around the world.

What Digital Immunoassays Can Learn from Ambient Analyte Theory: A Perspective.

Immunoassays are important tools for clinical diagnosis as well as environmental and food analysis because they enable highly sensitive and quantitative measurements of analyte concentrations. In the 1980s, Roger Ekins suggested to improve the sensitivity of immunoassays by employing microspot assays, which are carried out under ambient analyte conditions and do not change the bulk analyte concentration of a sample during a measurement. More recently, the measurement of single analyte molecules has additionally attracted wide research interest. Although the ability to detect a single analyte molecule is not synonymous with the highest analytical sensitivity, single-molecule detection makes new routes accessible to avoiding background noise. This perspective follows the development of solid-phase immunoassays from the design of label techniques to single-molecule (digital) assays against the backdrop of Ekins's fundamental work on immunoassay theory. The essential aspects of both ambient analyte and digital assay approaches are presented as a guideline to finding a balance between the speed, sensitivity, and precision of immunoassays.

Influence of Label and Solid Support on the Performance of Heterogeneous Immunoassays.

Conventional immunochemical methods used in clinical analysis are often not sensitive enough for early-stage diagnosis, resulting in the need for novel assay formats. Here, we provide a detailed comparison of the effect of different labels and solid supports on the performance of heterogeneous immunoassays. When comparing three types of streptavidin-modified labels─horseradish peroxidase, carboxyfluorescein, and photon-upconversion nanoparticles (UCNPs)─UCNPs led to the most sensitive and robust detection of the cancer biomarker prostate-specific antigen. Additionally, we compared the immunoassay formats based on conventional microtiter plates and magnetic microbeads (MBs). In both cases, the highest signal-to-background ratios and the lowest limits of detection (LODs) were obtained by using the UCNP labels. The MB-based upconversion-linked immunosorbent assay carried out with a preconcentration step provided the lowest LOD of 0.46 pg/mL in serum. The results demonstrate that the use of UCNPs and MBs can significantly improve the sensitivity and working range of heterogeneous immunoassays for biomarker detection.

Biosensing based on upconversion nanoparticles for food quality and safety applications.

Food safety and quality regulations inevitably call for sensitive and accurate analytical methods to detect harmful contaminants in food and to ensure safe food for the consumer. Both novel and well-established biorecognition elements, together with different transduction schemes, enable the simple and rapid analysis of various food contaminants. Upconversion nanoparticles (UCNPs) are inorganic nanocrystals that convert near-infrared light into shorter wavelength emission. This unique photophysical feature, along with narrow emission bandwidths and large anti-Stokes shift, render UCNPs excellent optical labels for biosensing because they can be detected without optical background interferences from the sample matrix. In this review, we show how this exciting technique has evolved into biosensing platforms for food quality and safety monitoring and highlight recent applications in the field.

Laser-induced breakdown spectroscopy as a readout method for immunocytochemistry with upconversion nanoparticles.

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.

Versatile Bioconjugation Strategies of PEG-Modified Upconversion Nanoparticles for Bioanalytical Applications.

Lanthanide-doped upconversion nanoparticles (UCNPs) display highly beneficial photophysical features for background-free bioimaging and bioanalysis; however, they are instable in high ionic strength buffers, have no functional groups, and are nonspecifically interacting. Here, we have prepared NIR-excitable UCNPs that are long-term colloidally stable in buffered media and possess functional groups. Heterobifunctional poly(ethylene glycol) (PEG) linkers bearing neridronate and alkyne or maleimide were attached to UCNPs via a ligand exchange. Streptavidin (SA)-conjugates were prepared by click reaction of UCNP@PEG-alkyne and SA-azide. Antihuman serum albumin pAbF antibody was modified with azide groups and conjugated to UCNP@PEG-alkyne via click reaction; alternatively, the antibody, after mild reduction of its disulfide bonds, was conjugated to UCNP@PEG-maleimide. We employed these nanoconjugates as labels for an upconversion-linked immunosorbent assay. SA-based labels achieved the lowest LOD of 0.17 ng/mL for the target albumin, which was superior compared to a fluorescence immunoassay (LOD 0.59 ng/mL) or an enzyme-linked immunoassay (LOD 0.56 ng/mL).

Advances in Optical Single-Molecule Detection: En†Route to Supersensitive Bioaffinity Assays.

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.

Large-Scale Purification of Photon-Upconversion Nanoparticles by Gel Electrophoresis for Analogue and Digital Bioassays.

The performance of photon-upconversion nanoparticles (UCNPs) as background-free luminescent labels in bioanalytical applications strongly depends on the preparation of well-defined and water-dispersible nanoconjugates. We have exploited the separation power of agarose-gel electrophoresis to purify milligram amounts of homogeneous UCNPs covered with carboxylated silica, biotin, or streptavidin with recovery rates of 30 to 50%. Clusters containing discrete numbers of UCNPs were isolated from the gel and reanalyzed by agarose-gel electrophoresis, single-nanoparticle-upconversion microscopy, and additional complementary methods. The purified nanoconjugates improved conventional (analogue) bioaffinity assays and provided highly monodisperse conjugates for assays that rely on counting individual UCNPs (digital assays).

Measurement of Sub-femtomolar Concentrations of Prostate-Specific Antigen through Single-Molecule Counting with an Upconversion-Linked Immunosorbent Assay.

Single-molecule (digital) immunoassays provide the ability to detect much lower protein concentrations than conventional immunoassays. As photon-upconversion nanoparticles (UCNPs) can be detected without optical background interference, they are excellent labels for so-called single-molecule upconversion-linked immunosorbent assays (ULISAs). We have introduced a UCNP label design based on streptavidin-PEG-neridronate and a two-step detection scheme involving a biotinylated antibody that efficiently reduces nonspecific binding on microtiter plates. In a microtiter plate immunoassay, individual sandwich immune complexes of the cancer marker prostate-specific antigen (PSA) are detected and counted by wide-field epiluminescence microscopy (digital readout). The digital detection is 16× more sensitive than the respective analogue readout and thus expands the limit of detection to the sub-femtomolar concentration range (LOD: 23 fg mL-1, 800 aM). The single molecule ULISA shows excellent correlation with an electrochemiluminescence reference method. Although the analogue readout can routinely measure PSA concentrations in human serum samples, very low concentrations have to be monitored after radical prostatectomy. Combining the digital and analogue readout covers a dynamic range of more than 3 orders of magnitude in a single experiment.

Development of photoswitchable inhibitors for ß-galactosidase.

Azobenzenes are of particular interest as a photochromic scaffold for biological applications because of their high fatigue resistance, their large geometrical change between extended (trans) and bent (cis) isomer, and their diverse synthetic accessibility. Despite their wide-spread use, there is no reported photochromic inhibitor of the well-investigated enzyme ß-galactosidase, which plays an important role for biochemistry and single molecule studies. Herein, we report the synthesis of photochromic competitive ß-galactosidase inhibitors based on the molecular structure of 2-phenylethyl ß-d-thiogalactoside (PETG) and 1-amino-1-deoxy-ß-d-galactose (ß-d-galactosylamine). The thermally highly stable PETG-based azobenzenes show excellent photochromic properties in polar solvents and moderate to high photostationary states (PSS). The optimized compound 37 is a strong competitive inhibitior of ß-galactosidase from Escherichia coli and its inhibition constant (Ki) changes between 60 nM and 290 nM upon irradiation with light. Additional docking experiments supported the observed structure-activity relationship.

Single Molecule Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive Detection of Diagnostic Biomarkers.

The ability to detect disease markers at the single molecule level promises the ultimate sensitivity in clinical diagnosis. Fluorescence-based single-molecule analysis, however, is limited by matrix interference and can only probe a very small detection volume, which is typically not suitable for real world analytical applications. We have developed a microtiter plate immunoassay for counting single molecules of the cancer marker prostate specific antigen (PSA) using photon-upconversion nanoparticles (UCNPs) as labels that can be detected without background fluorescence. Individual sandwich immunocomplexes consisting of (1) an anti-PSA antibody immobilized to the surface of a microtiter well, (2) PSA, and (3) an anti-PSA antibody-UCNP conjugate were counted under a wide-field epifluorescence microscope equipped with a 980 nm laser excitation source. The single-molecule (digital) upconversion-linked immunosorbent assay (ULISA) reaches a limit of detection of 1.2 pg mL-1 (42 fM) PSA in 25% blood serum, which is about ten times more sensitive than commercial ELISAs, and covers a dynamic range of three orders of magnitude. This upconversion detection mode has the potential to pave the way for a new generation of digital immunoassays.

Perspectives and challenges of photon-upconversion nanoparticles - Part II: bioanalytical applications.

In Part II of this review series on lanthanide-doped photon-upconversion nanoparticles (UCNPs), we present and critically discuss the performance and suitability of UCNPs as background-free luminescent reporters in bioimaging and bioanalytical applications. The preparation of a biocompatible nanoparticle surface is an integral step for all life - science-related applications. UCNPs have found their way into a large number of diagnostic platforms, homogeneous and heterogeneous assay formats, and sensor applications. Many bioanalytical detection schemes involve Förster resonance energy transfer (FRET), which is still debated for UCNPs and needs to be much improved. The need for dedicated and standardized instruments as well as recent studies on the dissolution and potential toxicity of UCNPs are addressed. Finally we outline future trends and challenges in the field of upconversion. Graphical Abstract Both synthesis / spectroscopy as well bioanalytical applications of UCNPs are driven by the COST Action CM1403 "The European Upconversion Network".

Perspectives and challenges of photon-upconversion nanoparticles - Part I: routes to brighter particles and quantitative spectroscopic studies.

Lanthanide-doped photon-upconversion nanoparticles (UCNPs) have been the focus of many research activities in materials and life sciences in the last 15 years because of their potential to convert light between different spectral regions and their unique photophysical properties. To fully exploit the application potential of these fascinating nanomaterials, a number of challenges have to be overcome, such as the low brightness, particularly of small UCNPs, and the reliable quantification of the excitation-power-density-dependent upconversion luminescence. In this series of critical reviews, recent developments in the design, synthesis, optical-spectroscopic characterization, and application of UCNPs are presented with special focus on bioanalysis and the life sciences. Here we guide the reader from the synthesis of UCNPs to different concepts to enhance their luminescence, including the required optical-spectroscopic assessment to quantify material performance; surface modification strategies and bioanalytical applications as well as selected examples of the use of UCNPs as reporters in different assay formats are addressed in part II. Future trends and challenges in the field of upconversion are discussed with special emphasis on UCNP synthesis and material characterization, particularly quantitative luminescence studies. Graphical Abstract Both synthesis and spectroscopy as well bioanalytical applications of UCNPs are driven and supported by COST Action CM1403 "The European Upconversion Network".

Competitive Upconversion-Linked Immunosorbent Assay for the Sensitive Detection of Diclofenac.

Photon-upconverting nanoparticles (UCNPs) emit light of shorter wavelength under near-infrared excitation and thus avoid optical background interference. We have exploited this unique photophysical feature to establish a sensitive competitive immunoassay for the detection of the pharmaceutical micropollutant diclofenac (DCF) in water. The so-called upconversion-linked immunosorbent assay (ULISA) was critically dependent on the design of the upconversion luminescent detection label. Silica-coated UCNPs (50 nm in diameter) exposing carboxyl groups on the surface were conjugated to a secondary anti-IgG antibody. We investigated the structure and monodispersity of the nanoconjugates in detail. Using a highly affine anti-DCF primary antibody, the optimized ULISA reached a detection limit of 0.05 ng DCF per mL. This performance came close to a conventional enzyme-linked immunosorbent assay (ELISA) without the need for an enzyme-mediated signal amplification step. The ULISA was further employed for analyzing drinking and surface water samples. The results were consistent with a conventional ELISA as well as liquid chromatography-mass spectrometry (LC-MS).

Highly Sensitive Laser Scanning of Photon-Upconverting Nanoparticles on a Macroscopic Scale.

An upconversion laser scanner has been optimized to exploit the advantages of photon-upconverting nanoparticles (UCNPs) for background-free imaging on a macroscopic scale. A collimated 980 nm laser beam afforded high local excitation densities to account for the nonlinear luminescence response of UCNPs. As few as 2000 nanoparticles were detectable, and the linear dynamic range covered more than 5 orders of magnitude, which is essentially impossible by using conventional fluorescent dyes. UCNPs covered by a dye-doped silica shell were separated by agarose gel electrophoresis and scanned by a conventional fluorescence scanner as well as the upconversion scanner. Both optical labels could be detected independently. Finally, upconversion images of lateral flow test strips were recorded to facilitate the sensitive and quantitative detection of disease markers. A marker for the parasitic worm Schistosoma was used in this study.

Surface modification and characterization of photon-upconverting nanoparticles for bioanalytical applications.

Photon-upconverting nanoparticles (UCNPs) can be excited by near-infrared light and emit visible light (anti-Stokes emission) which prevents autofluorescence and light scattering of biological samples. The potential for background-free imaging has attracted wide interest in UCNPs in recent years. Small and homogeneous lanthanide-doped UCNPs that display high upconversion efficiency have typically been synthesized in organic solvents. Bioanalytical applications, however, require a subsequent phase transfer to aqueous solutions. Hence, the surface properties of UCNPs must be well designed and characterized to grant both a stable aqueous colloidal dispersion and the ability to conjugate biomolecules and other ligands on the nanoparticle surface. In this review, we introduce various routes for the surface modification of UCNPs and critically discuss their advantages and disadvantages. The last part covers various analytical methods that enable a thorough examination of the progress and success of the surface functionalization.

Three-in-one enzyme assay based on single molecule detection in femtoliter arrays.

Large arrays of femtoliter-sized chambers are important tools for single molecule research as well as bioanalytical applications. We have optimized the design and fabrication of two array types consisting of 250 × 250 (62 500) femtoliter chambers either by surface etching of fused silica slides or by polydimethylsiloxane (PDMS) molding. Highly diluted solutions of ß-galactosidase were enclosed in such arrays to monitor the fluorogenic reactions of hundreds of individual enzyme molecules in parallel by wide-field fluorescence microscopy. An efficient mechanical sealing procedure was developed to prevent diffusion of the fluorescent reaction product out of the chambers. Different approaches for minimizing non-specific surface adsorption were explored. The signal acquisition was optimized to grant both a large field of view and an efficient signal acquisition from each femtoliter chamber. The optimized femtoliter array has enabled a three-in-one enzyme assay system: First, the concentration of active enzyme can be determined in a digital way by counting fluorescent chambers in the array. Second, the activity of the enzyme bulk solution is given by averaging many individual substrate turnover rates without the need for knowing the exact enzyme concentration. Third-unlike conventional enzyme assays-the distribution of individual substrate turnover rates yields insight into the conformational heterogeneity in an enzyme population. The substrate turnover rates of single ß-galactosidase molecules were found to be broadly distributed and independent of the type of femtoliter array. In general, both types of femtoliter arrays are highly sensitive platforms for enzyme analysis at the single molecule level and yield consistent results. Graphical Abstract Isolation and analysis of individual enzyme molecules in large arrays of femtoliter-sized chambers.

Special issue: single molecule techniques.

Technological advances in the detection and manipulation of single molecules have enabled new insights into the function, structure and interactions of biomolecules. This Special Issue was launched to account for the rapid progress in the field of "Single Molecule Techniques". Four original research articles and seven review articles provide an introduction, as well as an in-depth discussion, of technical developments that are indispensable for the characterization of individual biomolecules. Fluorescence microscopy takes center stage in this Special Issue because it is one of the most sensitive and flexible techniques, which has been adapted in many variations to the specific demands of single molecule analysis. Two additional articles are dedicated to single molecule detection based on atomic force microscopy.