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.

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.

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.

Nanoparticle-Based Bioaffinity Assays: From the Research Laboratory to the Market.

Advances in the development of new biorecognition elements, nanoparticle-based labels as well as instrumentation have inspired the design of new bioaffinity assays. This review critically discusses the potential of nanoparticles to replace current enzymatic or molecular labels in immunoassays and other bioaffinity assays. Successful implementations of nanoparticles in commercial assays and the need for rapid tests incorporating nanoparticles in different roles such as capture support, signal generation elements, and signal amplification systems are highlighted. The limited number of nanoparticles applied in current commercial assays can be explained by challenges associated with the analysis of real samples (e.g., blood, urine, or nasal swabs) that are difficult to resolve, particularly if the same performance can be achieved more easily by conventional labels. Lateral flow assays that are based on the visual detection of the red-colored line formed by colloidal gold are a notable exception, exemplified by SARS-CoV-2 rapid antigen tests that have moved from initial laboratory testing to widespread market adaption in less than two years.

High contrast breast cancer biomarker semi-quantification and immunohistochemistry imaging using upconverting nanoparticles.

Breast cancer is the second leading cause of cancer death in women. Current clinical treatment stratification practices open up an avenue for significant improvements, potentially through advancements in immunohistochemistry (IHC) assessments of biopsies. We report a high contrast upconverting nanoparticles (UCNP) labeling to distinguish different levels of human epidermal growth factor receptor 2 (HER2) in HER2 control pellet arrays (CPAs) and HER2-positive breast cancer tissue. A simple Fourier transform algorithm trained on CPAs was sufficient to provide a semi-quantitative HER2 assessment tool for breast cancer tissues. The UCNP labeling had a signal-to-background ratio of 40 compared to the negative control.

Extracellular Galectin 4 Drives Immune Evasion and Promotes T-cell Apoptosis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by rich deposits of extracellular matrix (ECM), affecting the pathophysiology of the disease. Here, we identified galectin 4 (gal 4) as a cancer cell-produced protein that was deposited into the ECM of PDAC tumors and detected high-circulating levels of gal 4 in patients with PDAC. In orthotopic transplantation experiments, we observed increased infiltration of T cells and prolonged survival in immunocompetent mice transplanted with cancer cells with reduced expression of gal 4. Increased survival was not observed in immunodeficient RAG1-/- mice, demonstrating that the effect was mediated by the adaptive immune system. By performing single-cell RNA-sequencing, we found that the myeloid compartment and cancer-associated fibroblast (CAF) subtypes were altered in the transplanted tumors. Reduced gal 4 expression associated with a higher proportion of myofibroblastic CAFs and reduced numbers of inflammatory CAFs. We also found higher proportions of M1 macrophages, T cells, and antigen-presenting dendritic cells in tumors with reduced gal 4 expression. Using a coculture system, we observed that extracellular gal 4 induced apoptosis in T cells by binding N-glycosylation residues on CD3ε/δ. Hence, we show that gal 4 is involved in immune evasion and identify gal 4 as a promising drug target for overcoming immunosuppression in PDAC.

Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging.

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.

Effect of Particle Size and Surface Chemistry of Photon-Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin.

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.

Enhanced resolution of generator-collector studies of enzymatic structures by means of hydrodynamic scanning electrochemical microscopy.

In this report, the effects of forced convection on scanning electrochemical microscopy (SECM) studies of enzymes in the context of the generator-collector mode (G/C mode) were investigated. Forced convection was generated via an electrical high precision stirrer integrated into the electrochemical cell. Circular spots of glucose oxidase were immobilized on a gold support serving as model substrate. The diffusion layer of enzymatically generated H2O2 was characterized recording probe scan curves (PSCs) in z-direction. Furthermore, the enzyme-modified surfaces were investigated via constant-height SECM imaging in feedback mode and in G/C mode. For methodical comparison all sets of experiments were performed in quiescent solution (conventional approach) and with forced convection, respectively. In contrast to a growing diffusion layer without forced convection by applying forced convection, a constant diffusion layer of produced H2O2 was observed. Hence, via hydrodynamic SECM time-independent images within a reasonable time scale of SECM measurements in G/C mode were enabled and their resolution was enhanced.

Surface design of photon-upconversion nanoparticles for high-contrast immunocytochemistry.

Immunohistochemistry (IHC) and immunocytochemistry (ICC) are routinely employed for the microscopic identification and diagnosis of cancerous cells in histological tissues and cell cultures. The maximally attainable contrast of conventional histological staining techniques, however, is low. While the anti-Stokes emission of photon-upconversion nanoparticles (UCNP) can efficiently eliminate optical background interference, excluding non-specific interactions of the label with the histological sample is equally important for specific immunolabeling. To address both requirements, we have designed and characterized several UCNP-based nanoconjugates as labels for the highly specific detection of the cancer biomarker HER2 on various breast cancer cell lines. An optimized streptavidin-PEG-neridronate-UCNP conjugate provided an unsurpassed signal-to-background ratio of 319, which was 50-fold better than conventional fluorescent labeling under the same experimental conditions. In combination, the absence of optical interference and non-specific binding lays the foundation for computer-based data evaluation in digital pathology.

Competitive upconversion-linked immunoassay using peptide mimetics for the detection of the mycotoxin zearalenone.

Due to increasing food safety standards, the analysis of mycotoxins has become essential in the food industry. In this work, we have developed a competitive upconversion-linked immunosorbent assay (ULISA) for the analysis of zearalenone (ZEA), one of the most frequently encountered mycotoxins in food worldwide. Instead of a toxin-conjugate conventionally used in competitive immunoassays, we designed a ZEA mimicking peptide extended by a biotin-linker and confirmed its excellent suitability to mimic ZEA by nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analysis. Upconversion nanoparticles (UCNP, type NaYF4:Yb,Tm) served as background-free optical label for the detection of the peptide mimetic in the competitive ULISA. Streptavidin-conjugated UCNPs were prepared by click reaction using an alkyne-PEG-neridronate linker. The UCNP conjugate clearly outperformed conventional labels such as enzymes or fluorescent dyes. With a limit of detection of 20 pg mL-1 (63 pM), the competitive ULISA is well applicable to the detection of ZEA at the levels set by the European legislation. Moreover, the ULISA is specific for ZEA and its metabolites (α- and ß-zearalenol) without significant cross-reactivity with other related mycotoxins. We detected ZEA in spiked and naturally contaminated maize samples using liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) as a reference method to demonstrate food analysis in real samples.

Click-conjugated photon-upconversion nanoparticles in an immunoassay for honeybee pathogen Melissococcus plutonius.

European foulbrood (EFB) is an infectious disease affecting honeybee larvae caused by the bacterium Melissococcus plutonius. The enzyme-linked immunosorbent assay (ELISA) is the gold standard for antibody-based bacteria detection, however, its sensitivity is not high enough to reveal early-stage EFB infection. Photon-upconversion nanoparticles (UCNPs) are lanthanide-doped nanomaterials that emit light of shorter wavelength under near-infrared (NIR) excitation and thus avoid optical background interference. After conjugation with specific biorecognition molecules, UCNPs can be used as ultrasensitive labels in immunoassays. Here, we introduce a method for conjugation of UCNPs with streptavidin based on copper-free click chemistry, which involves surface modification of UCNPs with alkyne-modified bovine serum albumin (BSA) that prevents the non-specific binding and provides reactive groups for conjugation with streptavidin-azide. To develop a sandwich upconversion-linked immunosorbent assay (ULISA) for M. plutonius detection, we have prepared a rabbit polyclonal anti-Melissococcus antibody. The specific capture of the bacteria was followed by binding of biotinylated antibody and UCNP-BSA-streptavidin conjugate for a highly sensitive upconversion readout. The assay yielded an LOD of 340 CFU mL-1 with a wide working range up to 109 CFU mL-1, which is 400 times better than the LOD of the conventional ELISA. The practical applicability of the ULISA was successfully demonstrated by detecting M. plutonius in spiked real samples of bees, larvae and bottom hive debris. These results show a great potential of the assay for early diagnosis of EFB, which can prevent uncontrolled spreading of the infection and losses of honeybee colonies.

Transition-State Ensembles Navigate the Pathways of Enzyme Catalysis.

Transition-state theory (TST) provides an important framework for analyzing and explaining the reaction rates of enzymes. TST, however, needs to account for protein dynamic effects and heterogeneities in enzyme catalysis. We have analyzed the reaction rates of ß-galactosidase and ß-glucuronidase at the single molecule level by using large arrays of femtoliter-sized chambers. Heterogeneities in individual reaction rates yield information on the intrinsic distribution of the free energy of activation (Δ G‡) in an enzyme ensemble. The broader distribution of Δ G‡ in ß-galactosidase compared to ß-glucuronidase is attributed to ß-galactosidase's multiple catalytic functions as a hydrolase and a transglycosylase. Based on the catalytic mechanism of ß-galactosidase, we show that transition-state ensembles do not only contribute to enzyme catalysis but can also channel the catalytic pathway to the formation of different products. We conclude that ß-galactosidase is an example of natural evolution, where a new catalytic pathway branches off from an established enzyme function. The functional division of work between enzymatic substates explains why the conformational space represented by the enzyme ensemble is larger than the conformational space that can be sampled by any given enzyme molecule during catalysis.