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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.

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.

American foulbrood (AFB) caused by the bacterium Paenibacillus larvae is the most destructive disease of the honeybee brood. Therefore, rapid and sensitive detection methods are required to limit spreading of this pathogen, which has a major impact on agriculture and biodiversity. While P. larvae is typically detected by microbial cultivation or polymerase chain reaction, antibody-based detection represents a viable alternative. Here, we prepared an antibody specific for P. larvae and used it for the development of an upconversion-linked immunosorbent assay (ULISA). Photon-upconversion nanoparticles (UCNP) were conjugated to streptavidin via a PEG-linker using copper-catalyzed click chemistry to replace the enzyme label in conventional enzyme-linked immunosorbent assay (ELISA). The ULISA showed low cross-reactivity and provided a limit of detection of 2.9 × 103 CFU/mL, representing a 22-fold improvement compared to the ELISA. This level is within the bacterial loads present in honeybee larvae during an AFB infection. The assay was successfully applied to the analysis of spiked samples of bees, larvae, and hive debris.

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).

Surface engineering of upconverting nanoparticles (UCNPs) is crucial for their bioanalytical applications. Here, an antibody specific to cardiac troponin I (cTnI), an important biomarker for acute myocardial infection, was covalently immobilized on the surface of UCNPs to prepare a label for the detection of cTnI biomarker in an upconversion-linked immunoassay (ULISA). Core-shell UCNPs (NaYF

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.

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.

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.

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 β-galactosidase, which plays an important role for biochemistry and single molecule studies. Herein, we report the synthesis of photochromic competitive β-galactosidase inhibitors based on the molecular structure of 2-phenylethyl β-d-thiogalactoside (PETG) and 1-amino-1-deoxy-β-d-galactose (β-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 β-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.

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".

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".

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.

The unique photoluminescent properties of upconversion nanoparticles (UCNPs) have attracted worldwide research interest and inspired many bioanalytical applications. The anti-Stokes emission with long luminescence lifetimes, narrow and multiple absorption and emission bands, and excellent photostability enable background-free and multiplexed detection in deep tissues. So far, however, in vitro and in vivo applications of UCNPs are restricted to the laboratory use due to safety concerns. Possible harmful effects may originate from the chemical composition but also from the small size of UCNPs. Potential end users must rely on well-founded safety data. Thus, a risk to benefit assessment of the envisioned combined therapeutic and diagnostic ("theranostic") applications is fundamentally important to bridge the translational gap between laboratory and clinics. The COST Action CM1403 "The European Upconversion Network-From the Design of Photon-Upconverting Nanomaterials to Biomedical Applications" integrates research on UCNPs ranging from fundamental materials synthesis and research, detection instrumentation, biofunctionalization, and bioassay development to toxicity testing. Such an interdisciplinary approach is necessary for a better and safer theranostic use of UCNPs. Here, the status of nanotoxicity research on UCNPs is compared to other nanomaterials, and routes for the translation of UCNPs into clinical applications are delineated.

International audience; The Conference and Spring School on Properties, Design and Applications of Upconversion Nanomaterials (UPCON) provides a new forum for all experts and newcomers in the field of upconversion research. On the occasion of the second UPCON 2018 in Valencia (Spain), we are pleased to present a collection of 12 reviews and research articles that reflect recent advances in upconversion materials, their unique luminescent properties and many applications spanning from nanoscale thermometry to biomedicine.

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).

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 β-galactosidase and β-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 (ΔG‡) in an enzyme ensemble. The broader distribution of ΔG‡ in β-galactosidase compared to β-glucuronidase is attributed to β-galactosidase’s multiple catalytic functions as a hydrolase and a transglycosylase. Based on the catalytic mechanism of β-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 β-galactosidase is an example of natural evolution, where a new catal...

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.

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, whic...

Photon-upconverting nanoparticles (UCNPs) have attracted much interest as a new class of luminescent label for the background-free detection in bioanalytical applications. UCNPs and other nanoparticles are commonly coated with a silica shell to improve their dispersibility and chemical stability in aqueous buffer and to incorporate functional groups for subsequent bioconjugation steps. The process of silica coating, however, is difficult to control without suitable analytical and preparative methods. Here, we have introduced agarose gel electrophoresis for the analysis and purification of silica-coated UCNPs. The silica shell can be doped with a fluorescent dye for direct detection in the gel without influencing the structure or electrophoretic mobility of the nanoparticles. The preparation of a bare silica shell by reverse microemulsion resulted in individual nanoparticles but also distinct aggregates that could be separated and isolated from the agarose gel. In contrast, the preparation of an ultrathin carboxylated silica shell yielded non-aggregated UCNPs only that could be directly used for protein conjugation. Agarose gel electrophoresis has also facilitated an efficient separation of protein-UCNP conjugates from excess reagents.

The activities of hundreds of single molecules of β-galactosidase were monitored in the presence of fluorogenic substrates and two strong binding inhibitors—D-galactal and N-p-bromobenzylamino-hydroxymethyl-cyclopentanetriol (NpBHC). The stochastic binding and release of the inhibitors to single β-galactosidase molecules was studied in both pre-steady state and steady state conditions. The effect of inhibition on enzyme activity is described and compared for both inhibitors. The inhibitor exchange rate and the substrate turnover rate were computed for individual enzyme molecules. These parameters are shown to be heterogeneous across the enzyme population. We demonstrate an inverse correlation between these parameters thus demonstrating that competitive inhibition is tightly coupled to the nature of the active site of individual enzyme molecules.

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).

Dual lifetime referencing (DLR) is introduced as a rapid and self-referenced method for measuring the concentration of a fluorescent analyte in solution. The fluorescent cancer chemotherapeutic doxorubicin was chosen as a medically relevant analyte and blended with a reference dye (Ru(dpp)(3)) that displays overlapping excitation and emission spectra. The relative contributions of the short-lived (nanoseconds) fluorescent analyte and the long-lived (microseconds) reference dye define the observed lifetime. Measuring this lifetime by both frequency-domain DLR and time-domain DLR yields similar analytical ranges and limits of detection (0.4 μM). To assess the matrix effect of medical samples, the standard addition method was employed to both modes of DLR. Urine was spiked with doxorubicin and recovery rates of ≥97% were obtained.

Long-wavelength absorbing labels that change their color and fluorescence upon conjugation to proteins and other biomolecules provide two critical advantages over the wealth of conventional amine-reactive labels. At first, the progress of the labeling reaction can be monitored continuously either visually or by spectrometry without prior purification. Then, the labeled biomolecule can be investigated with red or near-infrared light, which minimizes background interference in biological samples. These unique characteristics are met by a group of long-wavelength absorbing cyanine dyes carrying a reactive chloro substituent for nucleophilic substitution with primary amines, which is accompanied by a color change from green to blue. In addition to this so-called chameleon effect, the dyes display an increase in fluorescence during the labeling reaction. Despite their structural similarity, the reactivity of the dyes differs strongly. The fastest labeling kinetics is observed with dye S 0378 as its five-membered ring affords a stabilizing effect on the intermediate carbocation during an S(N)1-type of nucleophilic substitution. The reaction mechanism of the amine-reactive cyanine dyes provides a blueprint for the design of future long-wavelength absorbing chameleon dyes.

Die Kompartimentierung von Reaktionen in Femtoliter(fL)-Gefasen und die Integration von fL-Gefasen in Arrays verstarkt und beschleunigt nicht nur die chemische und biochemische Analyse, sondern fuhrt auch zu neuen wissenschaftlichen Methoden und Erkenntnissen. Dieser Aufsatz stellt die verschiedenen fL-Gefase und Array-Formate vor und lotet ihre Anwendungen fur den Nachweis und die Charakterisierung von biologisch bedeutsamen Analyten aus. Wenn fL- Arrays mit Analyten, Sonden oder Zellen bestuckt werden, konnen tausende analytischer Messungen parallel durchgefuhrt werden. Der Einschluss einzelner Enzymmolekule in fL-Arrays ermoglicht die gleichzeitig Analyse einer Vielzahl individueller Enzymmolekule. Neue Nanofabrikationstechniken und immer empfindlichere Nachweismethoden sind die treibende Kraft auf dem Gebiet der analytischen Chemie “im Femtoliter”. Unsere Ubersicht befasst sich insbesondere mit dem Fortschritt und den Herausforderungen auf dem Gebiet der analytischen Chemie im fL mit Beispielen sowohl aus der Grundlagenforschung als auch aus der angewandten Forschung.

Many individual horseradish peroxidase (HRP) molecules were isolated and observed simultaneously by fluorescence microscopy in an array of 50 000 femtoliter chambers chemically etched into the surface of a glass optical fiber bundle. The substrate turnovers of hundreds of individual HRP molecules were readily analyzed, and the large number of molecules observed provided excellent statistics. In contrast to other enzymes used for single-molecule studies, the rates of product formation in the femtoliter array were, on average, 10 times lower than in bulk solution. We attribute this phenomenon to the particular redox-reaction mechanism of HRP that involves two separate steps of product formation. HRP first oxidizes fluorogenic substrate molecules like Amplex Red to radical intermediates. Two radical molecules subsequently undergo an enzyme-independent dismutation reaction, the rate of which is decreased when confined to a femtoliter chamber resulting in less product. This two-step reaction mechanism of the widely used Amplex Red, as well as other fluorogenic substrates, is often overlooked. The mechanism not only affects single-molecule studies with HRP but also bulk reactions at low substrate turnover rates.

The notorious degradation susceptibility of peptides is a major obstacle to their use as medicinal drugs. Assays with which the stability of peptides in complex proteolytic environments can be determined are thus indispensable for peptide drug development. Herein, we describe a new peptide proteolysis assay that meets that demand. It unites the high-throughput capacity of heterogeneous with the well-defined kinetic characteristics of homogeneous assay formats and operates on the cleavage-caused loss of a detection handle. We have confirmed the assay's accuracy with well-defined model interactions and proved its high versatility and robustness with a representative application where we determined the half-lives of 375 different peptides in a crude intestinal protease preparation. With this reliable, reproducible, and efficient assay the enzyme kinetics of any peptide-protease interaction is accessible even for complex protease solutions.

Inhibition kinetics of single-β-galactosidase molecules with the slow-binding inhibitor d -galactal have been characterized by segregating individual enzyme molecules in an array of 50,000 ultra small reaction containers and observing substrate turnover changes with fluorescence microscopy. Inhibited and active states of β-galactosidase could be clearly distinguished, and the large array size provided very good statistics. With a pre-steady-state experiment, we demonstrated the stochastic character of inhibitor release, which obeys first-order kinetics. Under steady-state conditions, the quantitative detection of substrate turnover changes over long time periods revealed repeated inhibitor binding and release events, which are accompanied by conformational changes of the enzyme's catalytic site. We proved that the rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk-reaction kinetics.

Optical-fiber bundles have been employed as a versatile substrate for the fabrication of high-density microwell arrays. In this minireview, we discuss the application of optical-fiber-bundle arrays for a variety of biological problems. For genomics studies and microbial pathogen detection, individual beads have been functionalized with DNA probes and then loaded into the microwells. In addition, beads differentially responsive to vapors have been employed in an artificial olfaction system. Microwell arrays have also been loaded with living cells to monitor their individual response to biologically active compounds over long periods. Finally, the microwells have been sealed to enclose single enzyme molecules that can be used to measure individual molecule catalytic activity.

A ganglioside GM1 probe bearing a dark-red fluorescent dye at the sphingosine moiety of the molecule was prepared by a convenient one-pot synthesis. The labeled GM1 permitted the detection of the natural ganglioside GM1 ligand Escherichia coli heat-labile enterotoxin subunit B (EtxB) in picomole quantities on a solid support. When an epitope mapping of several ganglioside binding proteins and protein fragments was performed by screening a cellulose membrane-bound synthetic library of 64 16mer peptides with the new probe, several peptides displaying ganglioside GM1 affinity could be identified. We consider the labeled glycolipid described herein a versatile tool for manifold biochemical investigations.

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 β-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 β-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.

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., Open Access Komponente aus der Allianzlizenz

The mechanisms that drive the evolution of new enzyme activity have been investigated by comparing the kinetics of wild-type and in vitro evolved β-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62,500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wild-type GUS. The broader activity distribution indicates a functional division of work between individual molecules in a population of partially evolved enzymes that-as so-called generalists-are characterized by their promiscuous activity with many different substrates.

Photon-upconverting nanoparticles (UCNPs) are lanthanide-doped nanocrystals that emit visible light under near-infrared excitation (anti-Stokes emission). This unique optical property precludes background fluorescence and light scattering from biological materials. The emission of multiple and narrow emission lines is an additional hallmark of UCNPs that opens up new avenues for optical encoding. Distinct emission signatures can be obtained if the multiple emission of UCNPs is tuned by their dopant composition or by surface modification with dyes. Tuning the intensity of only one of the multiple emission lines and using another one as a constant reference signal enables the design of ratiometric codes that are resistant to fluctuations in absolute signal intensities. Combining several UCNPs each displaying a distinct set of emission lines expands the coding capacity exponentially and lays the foundation for highly multiplexed analyte detection. This Review highlights the potential of UCNPs for labeling and encoding biomolecules, microspheres, and even whole cells.

Photon-upconverting nanoparticles (UCNPs) are lanthanide-doped nanocrystals that emit visible light under near-infrared excitation (anti-Stokes emission). This unique optical property precludes background fluorescence and light scattering from biological materials. The emission of multiple and narrow emission lines is an additional hallmark of UCNPs that opens up new avenues for optical encoding. Distinct emission signatures can be obtained if the multiple emission of UCNPs is tuned by their dopant composition or by surface modification with dyes. Tuning the intensity of only one of the multiple emission lines and using another one as a constant reference signal enables the design of ratiometric codes that are resistant to fluctuations in absolute signal intensities. Combining several UCNPs each displaying a distinct set of emission lines expands the coding capacity exponentially and lays the foundation for highly multiplexed analyte detection. This Review highlights the potential of UCNPs for labeling and encoding biomolecules, microspheres, and even whole cells.

The proteolytic activity of renin is a key element in the regulation of blood pressure and a main target for inhibitor design. Currently, the activity of renin and its inhibitors is mainly analyzed using radioimmunoassays or FRET-substrates, which both have their limitations. Here, a novel kinetic assay is presented that combines the advantages of a homogeneous proteolytic reaction and a robust heterogeneous detection in a sandwich immunoassay format. The proteolysis in solution is not influenced by surface interactions and yields accurate kinetic values, while the specific detection of the cleavage products on a microtiter plate strongly reduces interference by concomitant substances and allows for a self-referenced signal readout. A new enzyme kinetic scheme for the inhibition of renin has been developed and validated by using the model inhibitor pepstatin. This kinetic analysis is amenable to parallelization for large-scale inhibitor screening. Furthermore, it can be easily adapted to inhibitors of other medically important proteases.

Photon upconverting nanoparticles (UCNPs) have become an important new class of optical labels. Their unique property of emitting visible light after photo-excitation with near-infrared radiation enables biological imaging without background interference or cell damage. Biological applications require UCNPs that are dispersible in water and allow the attachment of biomolecules. Oleic acid-coated UCNPs obtained by solvothermal synthesis were functionalized with both hydrophilic PEG and thiol-reactive maleimides, either by ligand exchange or by silanization. Three different types of maleimide-functionalized UCNPs were prepared and characterized by transmission electron microscopy, dynamic light scattering and Raman spectroscopy. Ligand exchange of oleic acid by maleimide?PEG?COOH yielded UCNPs that did not aggregate, were colloidally stable and reacted readily with proteins. Such luminescent labels are required for background-free imaging and many other bioanalytical applications.

Photon upconverting nanoparticles convert near-infrared into visible light (anti-Stokes emission), which strongly reduces the background of autofluorescence and light scattering in biological materials. Hexagonal NaYF(4) nanocrystals doped with Yb(3+) as the sensitizer and Er(3+)/Ho(3+)/Tm(3+) as the activator display at least two emission lines that respond differently to temperature changes. The ratio of the main emission line intensities enables a self-referenced optical readout of the temperature in the physiologically relevant range from 20 to 45 °C. Upconverting nanoparticles of the type NaYF(4):Yb, Er covered by an inactive shell of NaYF(4) are bright and allow for resolving temperature differences of less than 0.5 °C in the physiological range. The optical readout of this nanoparticle-based thermometer offers many options for imaging the two-dimensional distribution of temperature.

The sensitivity of antibody/hapten-based labeling systems is limited by the natural affinity ceiling of immunoglobulins. Breaking this limit by antibody engineering is difficult. We thus attempted a different approach and investigated if the so-called bridge effect, a corecognition of the linker present between hapten and carrier protein during antibody generation, can be utilized to improve the affinity of such labeling systems. The well-known haptens 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D) were equipped with various linkers, and the resulting affinity change of their cognate antibodies was analyzed by ELISA. Anti-2,4-DNP antibodies exhibited the best affinity to their hapten when it was combined with aminobutanoic acid or aminohexanoic acid. The affinity of anti-2,4-D antibodies could be enhanced even further with longer aliphatic spacers connected to the hapten. The affinity toward aminoundecanoic acid-2,4-D derivatives, for instance, was improved about 100-fold compared to 2,4-D alone and yielded detection limits as low as 100 amoles of analyte. As the effect occurred for all antibodies and haptens tested, it may be sensible to implement the bridge effect in future antibody/hapten-labeling systems in order to achieve the highest sensitivity possible.

The compartmentalization of reactions in femtoliter (fL) containers and integration of fL containers into arrays not only enhances and accelerates chemical and biochemical analysis but also leads to new scientific methods and insights. This review introduces various fL container and array formats and explores their applications for the detection and characterization of biologically relevant analytes. By loading analytes, sensing elements, or cells into fL arrays, one can perform thousands of analytical measurements in parallel. Confining single enzyme molecules in fL arrays enables one to analyze large numbers of individual enzyme molecules simultaneously in solution. New nanofabrication techniques and progressively more sensitive detection methods drive the field of fL analytical chemistry. This review focuses on the progress and challenges in the field of fL analytical chemistry with examples of both basic and applied research.

The gastrointestinal tract is a hostile biological environment, yet not all ingested materials are destroyed. The minute differences that determine whether a substance persists or is digested, liberated, adsorbed, excreted, or taken up are still poorly understood. Most attempts to investigate the events occurring during an orogastrointestinal passage rely on simplified in vitro systems where an analyte is exposed to artificial intestinal fluids. To closely mimic the events in the gastrointestinal tract, the exact intestinal fluid composition and the in vivo concentration of its constituents must be known. The widely used lavage procedures, however, dilute the intestinal fluids to an extent that precludes recalculation to the original concentrations. Thus, we developed procedures with which undiluted murine intestinal fluid can be harvested; determined the in vivo concentrations of the digestive enzymes trypsin, chymotrypsin, and elastase and the adsorbents mucin and immunoglobulin A in small intestinal fluid of fasted and unfasted female Balb/c mice; and identified chymotrypsin and immunoglobulin A as valid endogenous dilution markers for the recalculation of aqueous lavages. With these technologies and information at hand, more reliable investigations on the fate of allergens, pathogens, food, and anthropogenic xenobiotics in the gastrointestinal tract will be possible.

The compartmentalization of reactions in femtoliter (fL) containers and integration of fL containers into arrays not only enhances and accelerates chemical and biochemical analysis but also leads to new scientific methods and insights. This review introduces various fL container and array formats and explores their applications for the detection and characterization of biologically relevant analytes. By loading analytes, sensing elements, or cells into fL arrays, one can perform thousands of analytical measurements in parallel. Confining single enzyme molecules in fL arrays enables one to analyze large numbers of individual enzyme molecules simultaneously in solution. New nanofabrication techniques and progressively more sensitive detection methods drive the field of fL analytical chemistry. This review focuses on the progress and challenges in the field of fL analytical chemistry with examples of both basic and applied research.

Many bioanalytic and diagnostic procedures rely on labels with which the molecule of interest can be tracked in or discriminated from accompanying like substances. Herein, we describe a new labeling and detection system based on derivatives of 2,4-dichlorophenoxyacetic acid (2,4-D) and anti-2,4-D antibodies. The 2,4-D system is highly sensitive with a K(D) of 7 x 10(-11) M for the hapten-antibody pair, can be used on a large variety of biomolecules such as proteins, peptides, carbohydrates, and nucleic acids, is not hampered by endogenous backgrounds because 2,4-D is a xenobiotic, and is robust because 2,4-D is a very stable compound that withstands the conditions of most reactions usually performed on biomolecules. With this unique blend of properties, the 2,4-D system compares favorably with its rivals digoxigenin (DIG)/anti-DIG and biotin/(strept)avidin and provides an interesting and powerful tool in biomolecular labeling.

Individual enzyme molecules have been observed to possess discrete and different turnover rates due to the presence of long-lived activity states. These stable activity states are thought to result from different molecular conformations or post-translational modifications. The distributions in kinetic activity observed in previous studies were obtained from small numbers of single enzyme molecules. Due to this limitation, it has not been possible to fully characterize the different kinetic and equilibrium binding parameters of single enzyme molecules. In this paper, we analyze hundreds of single beta-galactosidase molecules simultaneously; using a high-density array of 50,000 fL-reaction chambers, we confirm the presence of long-lived kinetic states within a population of enzyme molecules. Our analysis has isolated the source of kinetic variability to kcat. The results explain the kinetic variability within enzyme molecule populations and offer a deeper understanding of the unique properties of single enzyme molecules. Gaining a more fundamental understanding of how individual enzyme molecules work within a population should provide insight into how they affect downstream biochemical processes. If the results reported here can be generalized to other enzymes, then the stochastic nature of individual enzyme molecule kinetics should have a substantial impact on the overall metabolic activity within a cell.

Optical-fiber bundles have been employed as a versatile substrate for the fabrication of high-density microwell arrays. In this minireview, we discuss the application of optical-fiber-bundle arrays for a variety of biological problems. For genomics studies and microbial pathogen detection, individual beads have been functionalized with DNA probes and then loaded into the microwells. In addition, beads differentially responsive to vapors have been employed in an artificial olfaction system. Microwell arrays have also been loaded with living cells to monitor their individual response to biologically active compounds over long periods. Finally, the microwells have been sealed to enclose single enzyme molecules that can be used to measure individual molecule catalytic activity.