Rapid time-resolved luminescence based screening of bacteria in urine with luminescence modulating biosensing phages.

Urinary tract infections (UTIs) are a common problem worldwide. The most prevalent causative pathogen of UTI is Escherichia coli, focus of this study. The current golden standard for detecting UTI is bacterial culture, creating a major workload for hospital laboratories - cost-effective and rapid mass screening of patient samples is needed. Here we present an alternative approach to screen patient samples with a single-step assay utilising time-resolved luminescence and luminescence modulating biosensing phages. Filamentous phage M13 was biopanned for binding luminescence quenching metal (copper) and further E. coli. The screening assay luminescence modulation was further enhanced by selecting right chemical environment for the functioning phage clones. Semi-specific interaction between phage, target bacteria and metal was detected by modulation in the signal of a weakly chelating, easily quenchable lanthanide complex. In the presence of the target pathogen, the phages collected quenching metal from solution to the bacterial surface changing the quenching effect on the lanthanide label and thus modulating the signal. Our method was compared with the bacterial culture data obtained from 70 patient samples. The developed proof-of-principle screening assay showed sensitivity and a specificity at the 90% mark when compared to culture method although some samples had high turbidity and even blood. The detection limit of E. coli was in the range of 1000-10 000 colony forming units/mL. Untreated urine sample was screened and time-resolved luminescence signal result was achieved within 10 min in a single incubation step.

STED-TEM Correlative Microscopy Leveraging Nanodiamonds as Intracellular Dual-Contrast Markers.

Development of fluorescent and electron dense markers is essential for the implementation of correlative light and electron microscopy, as dual-contrast landmarks are required to match the details in the multimodal images. Here, a novel method for correlative microscopy that utilizes fluorescent nanodiamonds (FNDs) as dual-contrast probes is reported. It is demonstrated how the FNDs can be used as dual-contrast labels-and together with automatic image registration tool SuperTomo, for precise image correlation-in high-resolution stimulated emission depletion (STED)/confocal and transmission electron microscopy (TEM) correlative microscopy experiments. It is shown how FNDs can be employed in experiments with both live and fixed cells as well as simple test samples. The fluorescence imaging can be performed either before TEM imaging or after, as the robust FNDs survive the TEM sample preparation and can be imaged with STED and other fluorescence microscopes directly on the TEM grids.

Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing.

Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L-1 in preprocessed serum samples by reducing nonspecific binding. The developed preprocessing step by affinity purification removed interfering compounds and improved the recovery of spiked TSH from serum. The sensitivity enhancement was achieved by stabilization of the protein corona of the nanoparticle bioconjugates and a spot-coated configuration of the active solid-phase that reduced sedimentation of the nanoparticle bioconjugates and their contact time with antibody-coated solid phase, thus making use of the higher association rate of specific binding due to high avidity nanoparticle bioconjugates. Graphical Abstract We were able to decrease the lowest limit of detection and increase sensitivity of TSH immunoassay using Eu(III)-nanoparticles. The improvement was achieved by decreasing binding time of nanoparticle bioconjugates by small capture area and fast circular rotation. Also, we applied a step to stabilize protein corona of the nanoparticles and a serum-preprocessing step with a structurally related antibody.

Kinetics of bioconjugate nanoparticle label binding in a sandwich-type immunoassay.

Nanoparticle labels have enhanced the performance of diagnostic, screening, and other measurement applications and hold further promise for more sensitive, precise, and cost-effective assay technologies. Nevertheless, a clear view of the biomolecular interactions on the molecular level is missing. Controlling the ratio of molecular recognition over undesired nonspecific adhesion is the key to improve biosensing with nanoparticles. To improve this ratio with an aim to disallow nonspecific binding, a more detailed perspective into the kinetic differences between the cases is needed. We present the application of two novel methods to determine complex binding kinetics of bioconjugate nanoparticles, interferometry, and force spectroscopy. Force spectroscopy is an atomic force microscopy technique and optical interferometry is a direct method to monitor reaction kinetics in second-hour timescale, both having steadily increasing importance in nanomedicine. The combination is perfectly suited for this purpose, due to the high sensitivity to detect binding events and the ability to investigate biological samples under physiological conditions. We have attached a single biofunctionalized nanoparticle to the outer tip apex and studied the binding behavior of the nanoparticle in a sandwich-type immunoassay using dynamic force spectroscopy in millisecond timescale. Utilization of the two novel methods allowed characterization of binding kinetics in a time range spanning from 50 ms to 4 h. These experiments allowed detection and demonstration of differences between specific and nonspecific binding. Most importantly, nonspecific binding of a nanoparticle was reduced at contact times below 100 ms with the solid-phase surface.

Fuzzy liquid analysis by an array of nonspecifically interacting reagents: the taste of fluorescence.

Complex or unknown liquid analysis requires extensive instrumentation and laboratory work; simple field devices usually have serious limitations in functionality, sensitivity, and applicability. This communication presents a novel, effective, and simple approach to fingerprinting liquids. The method is based on nonspecific interactions of the sample liquid, a long lifetime luminescent europium label, and various surface modulators in an array form that is readily converted to a field analysis µTAS system. As compared to existing e-nose or e-tongue techniques, the method is unique both in terms of sensitivity and usability, mainly due to the well-known unique properties of the europium label. This communication demonstrates the use of this new method in distinguishing different wines, waters, alcohols, and artificially modified berry juices.

High-throughput screening of colonization samples for methicillin-resistant Staphylococcus aureus.

BACKGROUND: We present here the first application of 2-photon excited fluorescence detection (TPX) technology for the direct screening of clinical colonization samples for methicillin-resistant Staphylococcus aureus (MRSA). METHODS: A total of 125 samples from 14 patients with previously identified MRSA carriage and 16 controls from low-prevalence settings were examined. RESULTS: The results were compared to those obtained by both standard phenotypic and molecular methods. In identifying MRSA carriers, i.e. persons with at least 1 MRSA positive colonization sample by standard methods, the sensitivity of the TPX technique was 100%, the specificity 78%, the positive predictive value 75%, and the negative predictive value 100%. The TPX assay sensitivity per colonization sample was 89%, the specificity 93%, the positive predictive value 84%, and the negative predictive value 95%. The median time for a true-positive test result was 3 h and 26 min; negative test results are available after 13 h. The assay capacity was 48 samples per test run. CONCLUSIONS: The TPX MRSA technique could provide early preliminary results for clinicians, while simultaneously functioning as a selective enrichment step for further conventional testing. Costs and workload associated with hospital infection control can be reduced using this high-throughput, point-of-care compatible methodology.

Detecting disease associated biomarkers by luminescence modulating phages.

Assessment of risk for a given disease and the diagnosis of diseases is often based on assays detecting biomarkers. Antibody-based biomarker-assays for diseases such as prostate cancer are often ambiguous and biomarker proteins are frequently also elevated for reasons that are unspecific. We have opted to use luminescence modulating phages for the analysis of known acute inflammatory response biomarker CRP (C-reactive protein) and biomarkers of prostate cancer in urine samples. Firstly, CRP was used to simulate the detection process in a controlled chemical environment. Secondly, we tried to classify more challenging lethal prostate cancer samples from control samples. Our unique method utilizes a special biopanning process in order to create special phages capable of capturing a dye necessary for detection and potential biomarkers. As the biomarker-molecules interfere with the phages, dye is repelled from the phage network resulting in an altered reporter luminescence. These changes can be observed with an absorbance reader and even with the naked eye. The simple method could present an alternative for screening of disease biomarkers. For prostate cancer urine samples, we achieved a sensitivity of 80% and specificity of 75% to detect Grade Group (GG) 4 and 5 prostate cancer.

Automated cell tracking using StarDist and TrackMate.

The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance, and wound healing. Therefore, the mechanisms governing cellular locomotion have been under intense scrutiny over the last 50 years. One of the main tools of this scrutiny is live-cell quantitative imaging, where researchers image cells over time to study their migration and quantitatively analyze their dynamics by tracking them using the recorded images. Despite the availability of computational tools, manual tracking remains widely used among researchers due to the difficulty setting up robust automated cell tracking and large-scale analysis. Here we provide a detailed analysis pipeline illustrating how the deep learning network StarDist can be combined with the popular tracking software TrackMate to perform 2D automated cell tracking and provide fully quantitative readouts. Our proposed protocol is compatible with both fluorescent and widefield images. It only requires freely available and open-source software (ZeroCostDL4Mic and Fiji), and does not require any coding knowledge from the users, making it a versatile and powerful tool for the field. We demonstrate this pipeline's usability by automatically tracking cancer cells and T cells using fluorescent and brightfield images. Importantly, we provide, as supplementary information, a detailed step-by-step protocol to allow researchers to implement it with their images.

Methicillin-resistant Staphylococcus aureus screening by online immunometric monitoring of bacterial growth under selective pressure.

Rapid, high-throughput screening tools are needed to contain the spread of hospital-acquired methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) strains. Most techniques used in current clinical practice still require time-consuming culture for primary isolation of the microbe. We present a new phenotypic assay for MRSA screening. The technique employs a two-photon excited fluorescence (TPX) detection technology with S. aureus-specific antibodies that allows the online monitoring of bacterial growth in a single separation-free process. Different progressions of fluorescence signals are recorded for methicillin-susceptible and -resistant strains when the growth of S. aureus is monitored in the presence of cefoxitin. The performance of the new technique was evaluated with 20 MRSA strains, 6 methicillin-susceptible S. aureus strains, and 7 coagulase-negative staphylococcal strains and two different monoclonal S. aureus-specific antibodies. When either of these antibodies was used, the sensitivity and the specificity of the TPX assay were 100%. All strains were correctly classified within 8 to 12 h, and up to 70 samples were simultaneously analyzed on a single 96-well microtiter plate. As a phenotypic method, the TPX assay is suited for screening purposes. The final definition of methicillin resistance in any S. aureus strain should be based on the presence of the mecA gene. The main benefit afforded by the initial use of the TPX methodology lies in its low cost and applicability to high-throughput analysis.

Intracellular Trafficking of Fluorescent Nanodiamonds and Regulation of Their Cellular Toxicity.

In this paper, cellular management of fluorescent nanodiamonds (FNDs) has been studied for better understanding in the design for potential applications of FNDs in biomedicine. The FNDs have shown to be photostable probes for bioimaging and thus are well-suited, for example, long-term tracking purposes. The FNDs also exhibit good biocompatibility and, in general, low toxicity for cell labeling. To demonstrate the underlying mechanism of cells coping the low but potentially toxic effects by nondegradable FNDs, we have studied their temporal intracellular trafficking. The FNDs were observed to be localized as distinct populations inside cells in early endosomes, lysosomes, and in proximity to the plasma membrane. The localization of FNDs in early endosomes suggests the internalization of FNDs, and lysosomal localization, in turn, can be interpreted as a prestate for exocytosis via lysosomal degradation pathway. The endocytosis and exocytosis appear to be occurring simultaneously in our observations. The mechanism of continuous endocytosis and exocytosis of FNDs could be necessary for cells to maintain normal proliferation. Furthermore, 120 h cell growth assay was performed to verify the long-term biocompatibility of FNDs for cellular studies.

Image Quality Ranking Method for Microscopy.

Automated analysis of microscope images is necessitated by the increased need for high-resolution follow up of events in time. Manually finding the right images to be analyzed, or eliminated from data analysis are common day-to-day problems in microscopy research today, and the constantly growing size of image datasets does not help the matter. We propose a simple method and a software tool for sorting images within a dataset, according to their relative quality. We demonstrate the applicability of our method in finding good quality images in a STED microscope sample preparation optimization image dataset. The results are validated by comparisons to subjective opinion scores, as well as five state-of-the-art blind image quality assessment methods. We also show how our method can be applied to eliminate useless out-of-focus images in a High-Content-Screening experiment. We further evaluate the ability of our image quality ranking method to detect out-of-focus images, by extensive simulations, and by comparing its performance against previously published, well-established microscopy autofocus metrics.

In vitro model of bone to facilitate measurement of adhesion forces and super-resolution imaging of osteoclasts.

To elucidate processes in the osteoclastic bone resorption, visualise resorption and related actin reorganisation, a combination of imaging technologies and an applicable in vitro model is needed. Nanosized bone powder from matching species is deposited on any biocompatible surface in order to form a thin, translucent, smooth and elastic representation of injured bone. Osteoclasts cultured on the layer expressed matching morphology to ones cultured on sawed cortical bone slices. Resorption pits were easily identified by reflectance microscopy. The coating allowed actin structures on the bone interface to be visualised with super-resolution microscopy along with a detailed interlinked actin networks and actin branching in conjunction with V-ATPase, dynamin and Arp2/3 at actin patches. Furthermore, we measured the timescale of an adaptive osteoclast adhesion to bone by force spectroscopy experiments on live osteoclasts with bone-coated AFM cantilevers. Utilising the in vitro model and the advanced imaging technologies we localised immunofluorescence signals in respect to bone with high precision and detected resorption at its early stages. Put together, our data supports a cyclic model for resorption in human osteoclasts.

Two-photon excitation in fluorescence polarization receptor-ligand binding assay.

Fluorescence polarization is one of the most commonly used homogeneous assay principles in drug discovery for screening of potential lead compounds. In this article, the fluorescence polarization technique is combined with 2-photon excitation of fluorescence. Theoretically, the use of 2-photon excitation of fluorescence increases the volumetric sensitivity and polarization contrast of fluorescence polarization assays. The work in this report demonstrates these predictions for an estrogen receptor ligand binding assay.

Functionalization of graphene oxide nanostructures improves photoluminescence and facilitates their use as optical probes in preclinical imaging.

Recently reported photoluminescent nanographene oxides (nGOs), i.e. nanographene oxidised with a sulfuric/nitric acid mixture (SNOx method), have tuneable photoluminescence and are scalable, simple and fast to produce optical probes. This material belongs to the vast class of photoluminescent carbon nanostructures, including carbon dots, nanodiamonds (NDs), graphene quantum dots (GQDs), all of which demonstrate a variety of properties that are attractive for biomedical imaging such as low toxicity and stable photoluminescence. In this study, the nGOs were organically surface-modified with poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) copolymers tagged with folic acid as the affinity ligand for cancer cells expressing folate receptors. The functionalization enhanced both the cellular uptake and quantum efficiency of the photoluminescence as compared to non-modified nGOs. The nGOs exhibited an excitation dependent photoluminescence that facilitated their detection with a wide range of microscope configurations. The functionalized nGOs were non-toxic, they were retained in the stained cell population over a period of 8 days and they were distributed equally between daughter cells. We have evaluated their applicability in in vitro and in vivo (chicken embryo CAM) models to visualize and track migratory cancer cells. The good biocompatibility and easy detection of the functionalized nGOs suggest that they could address the limitations faced with quantum dots and organic fluorophores in long-term in vivo biomedical imaging.

Detection methods of microsphere based single-step bioaffinity and in vitro diagnostics assays.

Microspheres provide a solid phase substrate for bioaffinity binding similar to the walls of traditional test tubes and the wells of microtiter plates. The coated microsphere concentrates analyte molecules in the reaction volume on its surface. When the bioaffinity binding reaction has reached an equilibrium, the local concentration of the analyte in close proximity of the microsphere is orders of magnitude higher than the concentration of the analyte in the total reaction volume. The preparation and quality control of microspheres coated with bioactive material is less costly and labour intensive when compared to test tube or microwell plate coating procedures. In addition, the cost for logistics and transportation of microsphere reagents is lower than that of coated tubes or plates. Moreover, microspheres can be easily used in miniaturised assay formats and several different detection schemes can be employed in the measurement of microsphere-based assays. Several different types of microspheres are commercially available. The microspheres can be manufactured in different sizes from many materials, such as polystyrene, acrylate, and glass. The surface of the microspheres can be activated to enable covalent binding of biomolecules. Further, the microspheres may contain internal fluorochrome or magnetic material, for identification or separation purposes. In this paper we review different assay formats for single-step measurement of bioaffinity assays employing microspheres. The term single-step is used to describe assays where all reagents and the sample are mixed, incubated and measured without separate washing steps.

Core-shell designs of photoluminescent nanodiamonds with porous silica coatings for bioimaging and drug delivery II: application.

Recent advances within materials science and its interdisciplinary applications in biomedicine have emphasized the potential of using a single multifunctional composite material for concurrent drug delivery and biomedical imaging. Here we present a novel composite material consisting of a photoluminescent nanodiamond (ND) core with a porous silica (SiO2) shell. This novel multifunctional probe serves as an alternative nanomaterial to address the existing problems with delivery and subsequent tracing of the particles. Whereas the unique optical properties of ND allows for long-term live cell imaging and tracking of cellular processes, mesoporous silica nanoparticles (MSNs) have proven to be efficient drug carriers. The advantages of both ND and MSNs were hereby integrated in the new composite material, ND@MSN. The optical properties provided by the ND core rendered the nanocomposite suitable for microscopy imaging in fluorescence and reflectance mode, as well as super-resolution microscopy as a STED label; whereas the porous silica coating provided efficient intracellular delivery capacity, especially in surface-functionalized form. This study serves as a demonstration how this novel nanomaterial can be exploited for both bioimaging and drug delivery for future theranostic applications.

Study on nonspecificity of an immuoassay using Eu-doped polystyrene nanoparticle labels.

Nanoparticle labels have been shown to improve the sensitivity of a sandwich immunoassay significantly. Further improvement in sensitivity is limited by nonspecific binding of the nanoparticle labels. Here, an experimental characterization of assay performance was carried out using clinically important analytes thyroid stimulating hormone and prostate-specific antigen. Particular attention was paid to characterization of nonspecific binding properties of nanoparticle labels. Therefore, different particle sizes and high affinity monoclonal antibodies (Mab) and their Fab and scFv recombinant antibody fragments were investigated. Combination of Fab fragment as a capture antibody and Mab as a detector antibody on a nanoparticle label resulted in high signal-to-background ratio consistently. Against the expectations no significant difference in nonspecific binding was found using fragmented antibodies compared to Mabs. The results also suggested that nonspecific binding was independent of the particle size. The particle size had a significant effect on the specific signal favouring the use of small particles giving a high specific signal. This study indicated that nonspecific binding is not readily affected by the physical size of the nanoparticle label or antibodies used in the assay.

Impact of surface defects and denaturation of capture surface proteins on nonspecific binding in immunoassays using antibody-coated polystyrene nanoparticle labels.

Microtiter wells are commonly used for bioassays. The sensitivity of such an assay depends on several instrumental and biochemical parameters such as the signal-to-background ratio and nonspecific binding of the label molecules. In this study, we have investigated the possible effects of well surface defects, well edges and denaturation of capture antibodies on the assay sensitivity. We used internally dyed Eu(III) chelate polystyrene nanoparticles as high specific activity labels in a thyroid stimulating hormone (TSH) sandwich-type model assay. The nanoparticle labels provide a high signal-to-background ratio in assays but the major limiting factor of the assay sensitivity is nonspecific binding of the labels. In our model assay the capture monoclonal antibodies were immobilized on microtiter wells passively or through streptavidin (SA)-biotin linkage. At first, commercially manufactured microtiter well surfaces were probed with an atomic force microscopy and significant structural inhomogeneities were found. The nonspecific binding of the nanoparticle conjugates did not appear to follow any of the microtiter well surface defect patterns in a number of experiments. In addition, the microtiter well edges did not increase the nonspecific binding. Denaturation of capture antibodies on solid surfaces has been proposed to expose amino acid sequences promoting nonspecific binding. This was studied by intentionally denaturing the surface capture antibodies by heat, detergent or acid treatment prior to the assay. Although specific signal was almost entirely lost no significant effect on nonspecific binding was observed. The passively adsorbed antibodies denatured at lower temperatures than those captured through streptavidin-biotin linkage. Evidently, the additional protein (SA) layer protected the capture antibody from denaturation whereas the solid surface appeared to act as a "catalyst" making the passively adsorbed antibody more susceptible to denaturation.

Rapid method for detection of influenza a and B virus antigens by use of a two-photon excitation assay technique and dry-chemistry reagents.

New separation-free assay methods for the rapid detection of influenza A and B virus antigens are presented. The methods employ dry-chemistry reagents and the recently developed two-photon excitation (TPX) fluorescence detection technology. According to the assay scheme, virus antigens are sandwiched by capture antibody onto polymer microspheres and fluorescently labeled antibody conjugate. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured, separation free, by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique for virus antigen detection, methods for influenza A and B viruses were constructed. The assay method for influenza A virus applied a molecular fluorescent label, whereas the method for influenza B virus required a nanoparticle fluorescent reporter to reach sufficient clinical sensitivity. The new methods utilize a dry-chemistry approach, where all assay-specific reagents are dispensed into assay wells already in the manufacturing process of the test kits. The performance of the assay methods was tested with nasopharyngeal specimens using a time-resolved fluoroimmunoassay as a reference method. The results suggest that the new technique enables the rapid detection of influenza virus antigens with sensitivity and specificity comparable to that of the reference method. The dose-response curves showed linear responses with slopes equal to unity and dynamic assay ranges of 3 orders of magnitude. Applicability of the novel TPX technique for rapid multianalyte testing of respiratory infections is discussed.