Time-resolved Fluorescence Detection Research Articles

Experimental triple-slit interference in a strongly driven V-type artificial atom

Rabi oscillations of a two-level atom appear as a quantum interference effect between the amplitudes associated to atomic superpositions, in analogy with the classic double-slit experiment which manifests a sinusoidal interference pattern. By extension, through direct detection of time-resolved resonance fluorescence from a quantum-dot neutral exciton driven in the Rabi regime, we experimentally demonstrate triple-slit-type quantum interference via quantum erasure in a V-type three-level artificial atom. This result is of fundamental interest in the experimental studies of the properties of V-type 3-level systems and may pave the way for further insight into their coherence properties as well as applications for quantum information schemes. It also suggests quantum dots as candidates for multi-path-interference experiments for probing foundational concepts in quantum physics.

High-sensitivity detection of cardiac troponin I with UV LED excitation for use in point-of-care immunoassay.

High-sensitivity cardiac troponin assay development enables determination of biological variation in healthy populations, more accurate interpretation of clinical results and points towards earlier diagnosis and rule-out of acute myocardial infarction. In this paper, we report on preliminary tests of an immunoassay analyzer employing an optimized LED excitation to measure on a standard troponin I and a novel research high-sensitivity troponin I assay. The limit of detection is improved by factor of 5 for standard troponin I and by factor of 3 for a research high-sensitivity troponin I assay, compared to the flash lamp excitation. The obtained limit of detection was 0.22 ng/L measured on plasma with the research high-sensitivity troponin I assay and 1.9 ng/L measured on tris-saline-azide buffer containing bovine serum albumin with the standard troponin I assay. We discuss the optimization of time-resolved detection of lanthanide fluorescence based on the time constants of the system and analyze the background and noise sources in a heterogeneous fluoroimmunoassay. We determine the limiting factors and their impact on the measurement performance. The suggested model can be generally applied to fluoroimmunoassays employing the dry-cup concept.

Conformational Control of Nitric Oxide Synthase Activity: Results from Time-Resolved and Single-Molecule Fluorescence

Nitric oxide synthase (NOS) generates a signaling molecule, nitric oxide, which is essential for processes such as vasodilation and neurotransmission. The enzyme forms homodimers containing cofactors FAD, FMN, and heme, each housed in subunits of the enzyme. Synthase activity in NOS is activated by calmodulin (CaM), which binds to a region adjacent to the FMN domain. Conformational motion is necessary to position sequential electron donor-acceptor pairs adjacent to one another for transfer of electrons from the FAD domain to the FMN domain and then to the oxygenase domain of the opposite member of the homodimer. We characterized the conformations and dynamics of endothelial and neuronal NOS with bound CaM by time-resolved and single-molecule detection of fluorescence from a fluorophore attached to CaM. Maximum entropy analysis of fluorescence decays for both neuronal and endothelial NOS reveals four conformational states, resolved by their distinct extents of quenching by energy transfer to heme. Clues to the identity of each population are found from their dependence on calcium concentration and from mutations known to disrupt specific interactions. The CaM mutation K115E, known to disrupt docking of CaM to the oxygenase domain, abolishes the two shortest lifetime populations. Single-molecule trajectories reveal conformational interchange on the millisecond to second time scales and permit analysis of the rates of interchange of conformational states. Exchange appears slowest to and from the conformation with highest quenching, consistent with the identification of this conformation as the “output” state of the enzyme.

A 365 nm UV LED-excitable antenna ligand for switchable lanthanide luminescence.

The time-resolved luminescence of lanthanide complexes is a highly sensitive and widely used bioassay technology for clinical diagnostics. With the time-resolved luminescence detection the naturally occurring autofluorescence of biological matrices, solid supports and plastics can be avoided. A major drawback of the current technique is that the luminescent lanthanide labels require ultraviolet (UV) excitation, typically shorter than 360 nm, which is strongly absorbed and can damage living biological systems. The lack of cost-efficient high power solid state excitation light sources for UV excitation further limits the development of low-cost and more compact measurement instruments for time-resolved luminescence and the potential use of lanthanide luminescence in different applications. Switchable lanthanide luminescence is a binary probe technology that inherently enables a high signal modulation in a separation-free detection of targets. The intrinsically luminescent lanthanide chelate is split into two nonluminescent moieties, a lanthanide ion carrier chelate and a light harvesting antenna ligand, each of which can be attached to a separate molecular probe. A luminescent lanthanide complex is formed only when the two probes bind adjacently to the target molecule. Herein we describe a new 365 nm excitable antenna ligand (AL360) for switchable lanthanide luminescence of europium(iii) (EuIII) that would enable the use of 365 nm light emitting diodes (LEDs) as an excitation light source for time-resolved fluorescence imaging and detection. With the acquired subpicomolar assay sensitivity it would be applicable for solution or surface arrays and UV LED microscopy.

Time-resolved Fluorescent Detection for Glucose Using a Complex of Luminescent Layered Titanates and Enzymes.

Luminescent europium-doped layered titanates (Eu-TiOx) were synthesized and complexed with horseradish peroxidase (HRP) and glucose oxidase (GOx). The emission of a resultant Eu-TiOx/HRP/GOx complex decreased upon the addition of glucose in the presence of guaiacol. The emission decrease was dependent on the concentrations of glucose, and the detection limit for glucose was 3.1 μM. The proposed system would be promising as a new detection method for glucose.

340 nm pulsed UV LED system for europium-based time-resolved fluorescence detection of immunoassays.

We report on the design, development and investigation of an optical system based on UV light emitting diode (LED) excitation at 340 nm for time-resolved fluorescence detection of immunoassays. The system was tested to measure cardiac marker Troponin I with a concentration of 200 ng/L in immunoassay. The signal-to-noise ratio was comparable to state-of-the-art Xenon flash lamp based unit with equal excitation energy and without overdriving the LED. We performed a comparative study of the flash lamp and the LED based system and discussed temporal, spatial, and spectral features of the LED excitation for time-resolved fluorimetry. Optimization of the suggested key parameters of the LED promises significant increase of the signal-to-noise ratio and hence of the sensitivity of immunoassay systems.

Automation Highlights from the Literature

Automation Highlights from the Literature

Lab-on-a-Drone: Toward Pinpoint Deployment of Smartphone-Enabled Nucleic Acid-Based Diagnostics for Mobile Health Care.

We introduce a portable biochemical analysis platform for rapid field deployment of nucleic acid-based diagnostics using consumer-class quadcopter drones. This approach exploits the ability to isothermally perform the polymerase chain reaction (PCR) with a single heater, enabling the system to be operated using standard 5 V USB sources that power mobile devices (via battery, solar, or hand crank action). Time-resolved fluorescence detection and quantification is achieved using a smartphone camera and integrated image analysis app. Standard sample preparation is enabled by leveraging the drone’s motors as centrifuges via 3D printed snap-on attachments. These advancements make it possible to build a complete DNA/RNA analysis system at a cost of ∼$50 ($US). Our instrument is rugged and versatile, enabling pinpoint deployment of sophisticated diagnostics to distributed field sites. This capability is demonstrated by successful in-flight replication of Staphylococcus aureus and λ-phage DNA targets in under 20 min. The ability to perform rapid in-flight assays with smartphone connectivity eliminates delays between sample collection and analysis so that test results can be delivered in minutes, suggesting new possibilities for drone-based systems to function in broader and more sophisticated roles beyond cargo transport and imaging.

Efficient signal processing for time-resolved fluorescence detection of nitrogen-vacancy spins in diamond

Room-temperature fluorescence detection of the nitrogen-vacancy center electronic spin typically has low signal to noise, requiring long experiments to reveal an averaged signal. Here, we present a simple approach to analysis of time-resolved fluorescence data that permits an improvement in measurement precision through signal processing alone. Applying our technique to experimental data reveals an improvement in signal to noise equivalent to a 14% increase in photon collection efficiency. We further explore the dependence of the signal to noise ratio on excitation power, and analyze our results using a rate equation model. Our results provide a rubric for optimizing fluorescence spin detection, which has direct implications for improving precision of nitrogen-vacancy-based sensors.

Cyclodextrin supramolecular inclusion-enhanced pyrene excimer switching for time-resolved fluorescence detection of biothiols in serum

We report here an efficient pyrene excimer signaling-based time-resolved fluorescent sensor for the measurement of biothiols (cysteine (Cys), homocysteine (Hcy), glutathione (GSH)) in human serum based on thymine–Hg2+–thymine (T–Hg2+–T) coordination chemistry and the inclusion interaction of cyclodextrin. The sensing mechanism of the approach is based on the competitive ligation of Hg2+ ions by Hcy/Cys/GSH and T–T mismatches in a bis-pyrene-labeled DNA strand with the self-complementary 5′ and 3′ ends. The introduction of γ-cyclodextrin can provide cooperation for the molecular level space proximity of the two labeled pyrene molecules, moreover the hydrophobic cavity of γ-cyclodextrin can also offer protection for the pyrene dimer's emission from the quenching effect of environmental conditions and enhance the fluorescence intensity of the pyrene excimer. When the biothiols are not presented, the sensing ensemble is in the “off” state due to the long distance between the two labeled pyrene molecules resulted from the formation of a more stable T–Hg2+–T structure. While in the presence of biothiols, Hg2+ interacts very strongly with thiol groups and the T–Hg2+–T structure is dehybridized, and then the pyrene excimer will be formed due to the self-complementary 5′ and 3′ ends of the DNA probe and the cooperation interaction of γ-cyclodextrin to pyrene dimer, thus resulting in switching the sensing ensemble to the “on” state. In the optimum conditions described, the linear concentration range of 1.0–100μM with the limit of detection (LOD) of 0.36μM for GSH was obtained. Moreover, due to the much longer lifetime of the pyrene excimer fluorescence than those of the ubiquitous endogenous fluorescent components, the time-resolved fluorescence technique has been successfully used for application in complicated biological samples.

Homogeneous time-resolved fluorescence assay for the detection of ricin using an aptamer immobilized on europium-doped KGdF4 nanoparticles and graphene oxide as a quencher

We have developed an aptamer-based assay for the food toxin ricin. It is based on a competitive quenching strategy along with time-resolved fluorescence detection. The assay involves the following steps: (a) The aptamer is immobilized on the europium-doped KGdF4 nanoparticles (NPs); (b) these NPs are added to the sample where ricin binds to the aptamer; (c) graphene oxide (GO) is added and competitively binds to the aptamer on the NPs that are not blocked by ricin; this causes the quenching of the fluorescence of the NPs; (d) fluorescence is detected at 593 nm in a microplate reader in the time-resolved mode at an excitation wavelength of 273 nm, a delay time of 100 μs, and a gating time of 1 s. Under optimal conditions, the calibration plot is linearly related to the concentration of ricin in the 50 pg·mL−1 to 50 ng·mL−1 range (R2 = 0.9975), and the limit of detection is 8 pg·mL−1. The method was compared to a standard ELISA, and correlation was excellent. The assay presented here provides a sensitive, dependable and convenient platform that is expected to have promising applications for the homogeneous assay of various other target analytes.

Out-of-equilibrium biomolecular interactions monitored by picosecond fluorescence in microfluidic droplets

We developed a new experimental approach combining Time-Resolved Fluorescence (TRF) spectroscopy and Droplet Microfluidics (DμF) to investigate the relaxation dynamics of structurally heterogeneous biomolecular systems. Here DμF was used to produce with minimal material consumption an out-of-equilibrium, fluorescently labeled biomolecular complex by rapid mixing within the droplets. TRF detection was implemented with a streak camera to monitor the time evolution of the structural heterogeneity of the complex along its relaxation towards equilibrium while it propagates inside the microfluidic channel. The approach was validated by investigating the fluorescence decay kinetics of a model interacting system of bovine serum albumin and Patent Blue V. Fluorescence decay kinetics are acquired with very good signal-to-noise ratio and allow for global, multicomponent fluorescence decay analysis, evidencing heterogeneous structural relaxation over several 100 ms.

SPAD Image Sensor With Analog Counting Pixel for Time-Resolved Fluorescence Detection

This paper presents a 32 × 32 pixel image sensor for time-gated fluorescence lifetime detection based on single-photon avalanche diodes. The sensor, fabricated in a high-voltage 0.35- μm CMOS technology, uses an analog counting approach to minimize the area occupation of pixel electronics while maintaining a nanosecond timing resolution and shot-noise-limited operation. The all nMOS pixel is formed by 12 transistors and features 25- μm pitch and 20.8% fill factor. The chip includes a phase-locked loop circuit for gating window generation, working at a maximum repetition frequency of 40 MHz, while the sensor can be gated at frequency up to 80 MHz using an external delay generator. Optical characterization with a picosecond-pulsed laser showed a minimum gating window width of 1.1 ns. Example images acquired in both continuous and time-gated mode are presented, together with a lifetime image obtained with the sensor mounted on a fluorescence microscope.

Label-Free Fluorescence Detection of Aromatic Compounds in Chip Electrophoresis Applying Two-Photon Excitation and Time-Correlated Single-Photon Counting

In this study, we introduce time-resolved fluorescence detection with two-photon excitation at 532 nm for label-free analyte determination in microchip electrophoresis. In the developed method, information about analyte fluorescence lifetimes is collected by time-correlated single-photon counting, improving reliable peak assignment in electrophoretic separations. The determined limits of detection for serotonin, propranolol, and tryptophan were 51, 37, and 280 nM, respectively, using microfluidic chips made of fused silica. Applying two-photon excitation microchip separations and label-free detection could also be performed in borosilicate glass chips demonstrating the potential for label-free fluorescence detection in non-UV-transparent devices. Microchip electrophoresis with two-photon excited fluorescence detection was then applied for analyses of active compounds in plant extracts. Harmala alkaloids present in methanolic plant extracts from Peganum harmala could be separated within seconds and detected with on-the-fly determination of fluorescence lifetimes.

Conformational Dynamics of a “Titratable” Cardiac Inotrope

Ischemic heart disease is a leading cause of morbidity and mortality in the world. To help gain insight into mechanisms and forge new experimental therapeutics we are using time-resolved fluorescence resonance energy transfer (TR-FRET) to detect structural changes within the cardiac troponin complex that occur during acute ischemic insult. During an ischemic event, penultimate to cell death, the sarcomere undergoes acidification. In cardiac myocytes, this decrease in pH uncouples calcium homeostasis from contractility. We have shown that the neonatal cardiac isoform of TnI, ssTnI, exhibits reduced pH-sensitivity compared to the adult cTnI isoform, however ssTnI confers deleterious effects at baseline in the context of an adult myocyte. Functional studies demonstrated that this pH-insensitivity stems from ssTnI residue H132. Introduction of a histidine at the homologous position in cTnI (A164H) mitigates the pH-sensitivity of the calcium-force relationship in cardiac myocytes. We are now establishing a TR-FRET approach to detect structural changes within the troponin complex in functional thin filaments with the goal of elucidating the structural dynamics that control pH-sensitive calcium-activated force production in the heart. We have engineered labeling sites designed to detect Ca2+ and pH sensitive structural changes in cTnI and cTnC. We will discuss our progress on utilizing a novel high-throughput fluorescence lifetime plate-reader that allows high-precision detection of time-resolved fluorescence.

Aptamer-directed lanthanide chelate self-assembly for rapid thrombin detection

We report a sensitive assay method for homogeneous thrombin detection. The method is based on lanthanide chelate complementation, where the luminescent complex is split into two separate label moieties, which are intrinsically non-luminescent. A luminescent mixed chelate complex is formed only when the label moieties are brought into close proximity directed by two separate binding events of aptamers to the analyte. This results in high specificity in signal generation while time-resolved fluorescence detection eliminates the short lifetime autofluorescence, which is inherent to many homogeneous assays and limits their applicability. The developed method is also very rapid as the maximum signal is obtained in just five minutes. Lanthanide chelate complementation can be applied for the detection of other proteins when two binders recognizing separate epitopes of the analyte are available.

Time-resolved fluorescent detection of Hg2+ in a complex environment by conjugating magnetic nanoparticles with a triple-helix molecular switch

In this communication, we molecularly engineered a light-switching excimer oligonucleotide-based probe for time-resolved fluorescent detection of Hg(2+). By combining a time-resolved fluorescence technique and magnetic nanoparticles, Hg(2+) spiked in human urine was detected.

Self-assembled Mn-doped ZnSe quantum dot–methyl viologen nanohybrids as an OFF–ON fluorescent probe for time-resolved fluorescence detection of tiopronin

The improvement of analytical accuracy and S/N (signal to noise ratio) based on conventional homogeneous fluorometry was always limited because of high background noise from complicated sample ingredients, buffer solution and lysates. Herein, a self-assembled Mn-doped ZnSe quantum dot–methyl viologen nanohybrid as an OFF–ON fluorescent probe is presented for the time-resolved fluorescence detection of tiopronin, showing high S/N and selectivity with a low detection limit of 0.1 μmol L−1 and a linear response ranging from 4 to 160 μM. We anticipate that this probe will have promising applications for sensitive biosensing and drug analysis.

Development of a dual functional luminescent sensor for zinc ion based on a peptidic architecture

A synthetic peptide bearing a lanthanide complex, TbOTZ exhibits a decrease of chromophore fluorescence and a concomitant luminescence enhancement due to sensitized Tb3+ upon Zn2+ binding. Thus, TbOTZ can be a valuable tool for ratiometric sensing of Zn2+ as well as for time-resolved fluorescence detection with a single molecule.

Multiwavelength time-resolved detection of fluorescence during the inflow of indocyanine green into the adult’s brain

Optical technique based on diffuse reflectance measurement combined with indocyanine green (ICG) bolus tracking is extensively tested as a method for clinical assessment of brain perfusion in adults at the bedside. Methodology of multiwavelength and time-resolved detection of fluorescence light excited in the ICG is presented and advantages of measurements at multiple wavelengths are discussed. Measurements were carried out: 1. on a physical homogeneous phantom to study the concentration dependence of the fluorescence signal, 2. on the phantom to simulate the dynamic inflow of ICG at different depths, and 3. in vivo on surface of the human head. Pattern of inflow and washout of ICG in the head of healthy volunteers after intravenous injection of the dye was observed for the first time with time-resolved instrumentation at multiple emission wavelengths. The multiwavelength detection of fluorescence signal confirms that at longer emission wavelengths, probability of reabsorption of the fluorescence light by the dye itself is reduced. Considering different light penetration depths at different wavelengths, and the pronounced reabsorption at longer wavelengths, the time-resolved multiwavelength technique may be useful in signal decomposition, leading to evaluation of extra- and intracerebral components of the measured signals.