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.

Advanced Cellulose Fibers for Efficient Immobilization of Enzymes.

Biocatalytic pulp fibers were prepared using surface functionalization of bleached kraft pulp with amino groups (F) and further immobilization of a cross-linked glucose oxidase (G*) from Aspergillus niger. The cross-linked enzymes (G*) were characterized using X-ray spectroscopy, Fourier transform infrared spectroscopy, dynamic scanning calorimetry, and dynamic light scattering. According to standard assays, the G* content on the resulting fibers (FG*) was of 11 mg/g of fiber, and enzyme activity was of 215 U/g. The results from confocal- and stimulated emission depletion microscopy techniques demonstrated that glucose oxidase do not penetrate the interlayers of fibers. The benefit of pulp fiber functionalization was evident in the present case, as the introduction of amino groups allowed the immobilization of larger amount of enzymes and rendered more efficient systems. Using the approach described on this paper, several advanced materials from wood pulp fibers and new bioprocesses might be developed by selecting the correct enzyme for the target applications.

Method for determination of polyethylene glycol molecular weight.

A method utilizing competitive adsorption between polyethylene glycols (PEGs) and labeled protein to nanoparticles was developed for the determination of PEG molecular weight (MW) in a microtiter plate format. Two mix-and-measure systems, time-resolved luminescence resonance energy transfer (TR-LRET) with donor europium(III) polystyrene nanoparticles and acceptor-labeled protein and quenching with quencher gold nanoparticles and fluorescently labeled protein were compared for their performance. MW is estimated from the PEG MW dependent changes in the competitive adsorption properties, which are presented as the luminescence signal vs PEG mass concentration. The curves obtained with the TR-LRET system overlapped for PEGs larger than 400 g/mol providing no information on MW. Distinctly different curves were obtained with the quenching system enabling the assessment of PEG MW within a broad dynamic range. The data was processed with and without prior knowledge of the PEG concentration to measure PEGs over a MW range from 62 to 35,000 g/mol. The demonstration of the measurement independent of the PEG concentration suggests that the estimation of MW is possible with quenching nanoparticle system for neutrally charged and relatively hydrophilic polymeric molecules widening the applicability of the simple and cost-effective nanoparticle-based methods.

Nanometric features of myosin filaments extracted from a single muscle fiber to uncover the mechanisms underlying organized motility.

The single muscle fiber in vitro motility assay (SF-IVMA) is characterized by organized linear motility of actin filaments, i.e., actin filaments motility showing a parallel or anti-parallel direction with similar speed independent of direction in the central part of the flow-cell where density of myosin is high. In contrast, the low myosin density region in the flow-cell exhibits random filament movements, but the mechanisms underlying the organized motility remain unknown. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) imaging techniques have been combined to investigate the morphological features of myosin extracted from single muscle fiber segments in the flow cell. Nanometric scale imaging of myosin filaments in the SF-IVMA showed intact spatial distances between myosin heads being essential for myosin filament function. However, angular spectrum analyses of myosin filaments in the high myosin density region showed organized myosin filament orientation only in small areas, while unorganized filament orientation were dominantly presented when larger areas were analyzed. Thus, parallel myosin filament organization is a less likely mechanism underlying the organized motility of actin filaments and the high myosin density per se is therefore forwarded as the primary "driver" that promotes organized linear motility.

Time-resolved fluorescence-based assay for rapid detection of Escherichia coli.

Fast and simple detection of pathogens is of utmost importance in health care and the food industry. In this article, a novel technology for the detection of pathogenic bacteria is presented. The technology uses lytic-specific bacteriophages and a nonspecific interaction of cellular components with a luminescent lanthanide chelate. As a proof of principle, Escherichia coli-specific T4 bacteriophage was used to infect the bacteria, and the cell lysis was detected. In the absence of E. coli, luminescent Eu(3+)-chelate complex cannot be formed and low time-resolved luminescence signal is monitored. In the presence of E. coli, increased luminescence signal is observed as the cellular contents are leached to the surrounding medium. The luminescence signal is observed as a function of the number of bacteria in the sample. The homogeneous assay can detect living E. coli in bacterial cultures and simulated urine samples within 25 min with a detection limit of 1000 or 10,000 bacterial cells/ml in buffer or urine, respectively. The detection limit is at the clinically relevant level, which indicates that the method could also be applicable to clinical settings for fast detection of urine bacteria.

Simple nanoparticle-based luminometric method for molecular weight determination of polymeric compounds.

A nanoparticle-based method utilizing time-resolved luminescence resonance energy transfer (TR-LRET) was developed for molecular weight determination. This mix-and-measure nanoparticle method is based on the competitive adsorption between the analyte and the acceptor-labeled protein to donor Eu(III) nanoparticles. The size-dependent adsorption of molecules enables the molecular weight determination of differently sized polymeric compounds down to a concentration level of micrograms per liter. The molecular weight determination from 1 to 10 kDa for polyamino acids and from 0.3 to 70 kDa for polyethylene imines is demonstrated. The simple and cost-effective nanoparticle method as microtiter plate assay format shows great potential for the detection of the changes in molecular weight or for quantification of differently sized molecules in biochemical laboratories and in industrial polymeric processes.

Non-competitive aptamer-based quenching resonance energy transfer assay for homogeneous growth factor quantification.

A non-competitive homogeneous, single-label quenching resonance energy transfer (QRET) assay for protein quantification is now presented using lanthanide-chelate labeled nucleic acid aptamers. A labeled ssDNA aptamer binding to a growth factor has been successfully used to provide luminescence signal protection of the lanthanide label. The QRET technology has previously been applied to competitive assay formats, but now for the first time a direct non-competitive assay is presented. The QRET system is based on the protection of the Eu(iii)-chelate from a soluble quencher molecule when the aptamer interacts with a specific target protein. The direct QRET assay is possible as the aptamer structure itself cannot protect the Eu(iii)-label from quenching. The dynamic range for the optimized vascular endothelial growth factor (VEGF) assay is 0.25-10 nM. A successful quantification of the basic fibroblast growth factor (bFGF) is also demonstrated using the same QRET assay format with a dynamic range of 0.75-50 nM. These assays evidently show the suitability of the direct QRET technique to simple and efficient detection of large biomolecules. The QRET assay can potentially be applied as a detection platform for any other protein targets with a known aptamer sequence.

A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction.

A quenching resonance energy transfer (QRET) assay for small GTPase nucleotide exchange kinetic monitoring is demonstrated using nanomolar protein concentrations. Small GTPases are central signaling proteins in all eukaryotic cells acting as a "molecular switches" that are active in the GTP-state and inactive in the GDP-state. GTP-loading is highly regulated by guanine nucleotide exchange factors (GEFs). In several diseases, most prominently cancer, this process in misregulated. The kinetics of the nucleotide exchange reaction reports on the enzymatic activity of the GEF reaction system and is, therefore, of special interest. We determined the nucleotide exchange kinetics using europium-labeled GTP (Eu-GTP) in the QRET assay for small GTPases. After GEF catalyzed GTP-loading of a GTPase, a high time-resolved luminescence signal was found to be associated with GTPase bound Eu-GTP, whereas the non-bound Eu-GTP fraction was quenched by soluble quencher. The association kinetics of the Eu-GTP was measured after GEF addition, whereas the dissociation kinetics could be determined after addition of unlabeled GTP. The resulting association and dissociation rates were in agreement with previously published values for H-Ras(Wt), H-Ras(Q61G), and K-Ras(Wt), respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoA(Wt) GTP-loading. The QRET assay allows the use of nanomolar protein concentrations, as more than 3-fold signal-to-background ratio was achieved with 50 nM GTPase and GEF proteins. Thus, small GTPase exchange kinetics can be efficiently determined in a HTS compatible 384-well plate format.

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.

Phage Biosensor for the Classification of Metastatic Urological Cancers from Urine.

Most of the annual 10 million cancer-related deaths are caused by metastatic disease. Survival rates for cancer are strongly dependent on the type of cancer and its stage at detection. Early detection remains a challenge due to the lack of reliable biomarkers and cost-efficient screening methods. Phage biosensors can offer a solution for early detection using non-invasive liquid biopsies. Here, we report the first results of the bifunctional phage biosensor to detect metastatic urological cancers from urine. A dye-sensitized phage library was used to select metastasis-related phage binders. After selection rounds, the most promising phage candidate was used to classify metastatic cancer from controls. Additionally, we applied one chemical sensor (phenoxazine non-fluorescent dye) to classify cancer from urine. A statistical significance (p = 0.0002) was observed between metastatic and non-metastatic cancer, with sensitivity of 70% and specificity of 79%. Furthermore, the chemical sensor demonstrated significance in detecting cancer (p < 0.0001) with a sensitivity of 71% and a specificity of 75%. Our data suggest a new promising field for urine biomarker research, and further evaluation with prospectively collected samples is ongoing. In conclusion, we report, for the first time, the potential of a chemical- and phage-based biosensor method to detect metastatic cancer using urine.

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.

Nonspecific particle-based method with two-photon excitation detection for sensitive protein quantification and cell counting.

A novel easy-to-use homogeneous method utilizing two-photon excitation (TPX) for quantification of proteins or counting of eukaryotic cells in solution has been developed. This highly sensitive technique is based on the adsorption competition between the sample and fluorescently labeled protein to micrometer-sized carboxylate modified polystyrene particles and detection of two-photon excited fluorescence. The adsorption of the labeled protein to the particles was detected as a distinct fluorescence on individual microparticles. Analyte protein or eukaryotic cells interacted with particle surface and reduced the adsorption of labeled protein to the particles resulting in a decrease of the fluorescence. The optimizations of assay conditions were performed separately for protein quantification and cell counting, and the principle of the method was confirmed with the fluorescence microscopy imaging. The protein quantification assay allowed the determination of picogram quantities (1.2 µg/L) of protein, and the cell counting assay allowed three cells in the sample with an average variation of approximately 10% in the signal. The protein assay sensitivity was more than 500-fold improved from the common most sensitive commercial methods. Moreover, the dynamic range of the assay was broad, approximately 4 orders of magnitude. The cell assay has sensitivity comparable to the most sensitive commercial method. The developed method tolerates interfering agents such as neutral detergents found in cell lysate samples even at high concentrations. The method is experimentally fairly simple and allows the expansion for the use of the TPX technology.

Protein quantification using resonance energy transfer between donor nanoparticles and acceptor quantum dots.

A homogeneous time-resolved luminescence resonance energy transfer (TR-LRET) assay has been developed to quantify proteins. The competitive assay is based on resonance energy transfer (RET) between two luminescent nanosized particles. Polystyrene nanoparticles loaded with Eu(3+) chelates (EuNPs) act as donors, while protein-coated quantum dots (QDs), either CdSe/ZnS emitting at 655 nm (QD655-strep) or CdSeTe/ZnS with emission wavelength at 705 nm (QD705-strep), are acceptors. In the absence of analyte protein, in our case bovine serum albumin (BSA), the protein-coated QDs bind nonspecifically to the EuNPs, leading to RET. In the presence of analyte proteins, the binding of the QDs to the EuNPs is prevented and the RET signal decreases. RET from the EuNPs to the QDs was confirmed and characterized with steady-state and time-resolved luminescence spectroscopy. In accordance with the Förster theory, the approximate average donor-acceptor distance is around 15 nm at RET efficiencies, equal to 15% for QD655 and 13% for QD705 acceptor, respectively. The limits of detection are below 10 ng of BSA with less than a 10% average coefficient of variation. The assay sensitivity is improved, when compared to the most sensitive commercial methods. The presented mix-and-measure method has potential to be implemented into routine protein quantification in biological laboratories.

Multiparametric homogeneous method for identification of ligand binding to G protein-coupled receptors: receptor-ligand binding and ß-arrestin assay.

Two homogeneous assay systems have been combined to provide a new cell-based functional assay. The assay can be used to identify ligand binding to ß(2)-adrenergic receptors, but also the downstream response can be determined in the same assay. Both the quenching resonance energy transfer (QRET) and the DiscoveRx PathHunter assay formats allow the use of intact cells. The homogeneous QRET technique is a single-label approach based on nonspecific quenching of the time-resolved luminescence, enabling agonist and antagonist receptor binding measurements. The commercial PathHunter assay is in turn based on enzyme fragment complementation, which can be detected on the basis of chemiluminescence signal. In the PathHunter technology the enzyme complementation is recorded immediately downstream of agonist-induced receptor activation. The new multiparametric detection technology combines these two assay methods enabling the identification of agonist, and antagonist binding to the receptor, and the agonist-induced response. Using the QRET and the PathHunter methods a panel of ß(2)-adrenergic receptor ligands (epinephrine, terbutaline, metaproterenol, salmeterol, propranolol, alprenolol, bisoprolol, ICI 118,551, and bucindolol) was tested to prove the assay performance. The signal-to-background ratio for tested ligands ranged from 5 to 11 and from 6 to 18 with QRET and PathHunter, respectively. Combined homogeneous assay technique can provide an informative method for screening purposes and an efficient way to monitor receptor-ligand interaction, thus separating agonist from antagonist.

Luminescence stability improvement in liposome-based homogeneous luminescence resonance energy transfer.

A stable liposome-based time-resolved luminescence resonance energy transfer (TR-LRET) assay was developed based on the interaction of biotinylated lipids and streptavidin. Eu(3+) ion chelated to 4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione and trioctylphosphine oxide was incorporated into liposomes. Acceptor-labeled streptavidin bound to biotinylated lipids of the liposomes enables TR-LRET. A stable assay performance was achieved by optimization. High Eu(3+) signal and stability, low variation, and sensitivity below 100 pM for free biotin was achieved by incorporating the chelate into liposomes containing cholesterol in a carbonate buffer. Potentially, the stable assay compared with the assay without cholesterol offers an improved platform to liposome-based detection systems.

A homogeneous single-label quenching resonance energy transfer assay for a δ-opioid receptor-ligand using intact cells.

This study, a homogeneous assay system for delta opioid receptor binding ligands has been developed using Quenching Resonance Energy Transfer (QRET). The QRET system allows receptor-ligand binding assays on intact cells using a single-label approach and a nonspecific quenching mechanism. Binding of antagonists or agonists to the receptor can be defined using a europium(III) labeled ligand. In the presence of the unlabeled ligand the labeled ligand is displaced and remains in solution. The non-bound labeled ligand is not protected by the target receptor, and the luminescence signal is quenched. For this objective, a Eu(III) labeled peptide molecule with three different linkers (AX0, AX1 and AX2) was designed. Peptides were evaluated using the homogeneous QRET technique, radioligand binding assays and the heterogeneous time-resolved luminescence (TRL) technique. Using the Eu-AX0 peptide and the QRET method, a panel of opioid compounds (naltrexone, naltrindole, SCN-80, DPDPE and DAMGO) was tested to prove the assay performance. The signal-to-background ratio for the tested opioid ligand ranged from 3.3 to 12.0. The QRET method showed prominent performance also in high DMSO concentrations. QRET is a homogenous and a non-radioactive detection system for screening and this is the first attempt to utilize peptide ligands in the QRET concept.

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.

Biophysical Properties of Bifunctional Phage-Biosensor.

Biosensor research is a swiftly growing field for developing rapid and precise analytical devices for biomedical, pharmaceutical, and industrial use and beyond. Herein, we propose a phage-based biosensor method to develop a sensitive and specific system for biomedical detection. Our method is based on in vitro selected phages and their interaction with the targeted analytes as well as on optical properties that change according to the concentration of the model analyte. The green fluorescent protein (GFP) was chosen as our model analyte as it has its own well-known optical properties. Brilliant green was used as a reporter component for the sensor. Its presence enables a color intensity (absorbance) change when the analyte is present in the solution. Furthermore, the reporter dye functioned as a quencher for an additional lanthanide label in our assay. It mediated the specific phage-derived interference in the signal measured with the time-resolved luminescence. Most importantly, our results confirmed that the presented bifunctional phage with its liquid crystal properties enabled the measurement of GFP in a concentration-dependent, quantitative manner with a limit of detection of 0.24 µg/mL. In the future, our novel method to develop phage-based biosensors may provide highly sensitive and specific biosensors for biomedical or otherwise-relevant targets.