Time-resolved Luminescence Detection Research Articles

Sensitive and High-Throughput Time-Resolved Luminescence Detection of Tetracycline in Milk for Eliminating Background Fluorescence on a Miniaturized Apparatus.

Fluorescence detection has always suffered from high background fluorescence from real samples such as milk. Therefore, cumbersome pretreatments of samples were necessary to remove the fluorescent substances but led to long processing times and low efficiency. Time-resolved luminescence detection is a powerful technique for eliminating short-lived background fluorescence without additional pretreatments. However, the related instruments are usually equipped with high-speed excitation sources and detectors, which are always bulky and expensive. Herein, we developed a low-cost and miniaturized imaging system for high-throughput time-gated luminescence detection. An UV LED array was used to excite multiple samples, the luminescence of which could be detected by a smartphone simultaneously. An analog circuit was designed to synchronize the LED to the mechanical chopper to eliminate the background signals resulting from scattering and short-lived autofluorescence. Compared to other synchronous circuits based on FPGAs and microcontrollers, this analog circuit required no programming and memory. For the first time, high-throughput time-resolved luminescence detection of tetracycline in milk without any separation or enrichment was achieved by utilizing a smartphone as a camera, and the scattered signals and the background fluorescence were eliminated efficiently. The limit of detection reached as low as 53 nM (∼0.024 ppm), lower than the residue limit set by the European Union. This high-throughput time-gated luminescence detection method can be used for quantitative analysis of many real samples with high background fluorescence.

Design and synthesis of Lanthanide(III) complex based luminescence probes for time-resolved luminescence detection of β-galactosidase

β-Galactosidase (β-Gal), confirmed to provide energy and participate in the galactosyl transfer reaction, is primarily over-expressed in ovarian cancer cells. Herein, detecting endogenous β-Gal is essential for the diagnosis and monitoring of primary ovarian cancer cells. In this study, we proposed two structurally related cationic Lanthanide(III) triamide complexes for highly sensitive and rapid detection of β-Gal. Moreover, LDEu02 showed a low detection limit (2.3 × 10−3 U/mL) and high specificity with high affinity for β-Gal, which had great potential to the detection of the β-Gal activity by using time-resolved fluorescence techniques.

A simple synthesis method of microsphere immunochromatographic test strip for time-resolved luminescence detection of folic acid

Folic acid (FA) is an ingredient that must be added to infant milk powder to avoid potential defects. Rapid, sensitive and reliable detection methods are needed to determined FA addition levels. Thus, this study established a microsphere immunochromatographic test strip for time-resolved luminescence detection (TRLM-ICTS) based on carboxyl-functionalized time-resolved luminescent microspheres (Eu-TRLMs) prepared by a one-step method as fluorescent markers for the immediate quantitative detection of FA in milk powder. Eu-TRLMs prepared by the one-step method showed good dispersion, high stability and strong fluorescence intensity, which is improving the sensitivity of TRLM-ICTS. In the performance evaluation of TRLM-ICTS, the detection limit was 0.487 ng mL−1, the recovery rate was 97.3–105 %, and the actual sample detection results were in line with those of UPLC-MS/MS. TRLM-ICTS has the advantages of rapid, high sensitivity and strong specificity and could as a practical quantitative detection method for the detection of FA in milk powder.

A high-nuclearity Cd(II)-Tb(III) nanocage for the rapid and quantitative time-resolved luminescence detection of guanosine-5-monophoshpate and RNA

One 32-metal Cd(II)-Tb(III) nanocage 1 was obtained using a flexible Schiff base ligand. The addition of guanosine-5-monophosphate (GMP) and RNA of human pancreas cancer results in the Tb(III) luminescence (5D4 → 7Fn transitions, n = 6, 5, 4 and 3), which is dominated by the static enhancement model. 1 exhibits highly sensitive time-resolved luminescence (TRL) detection to GMP and RNA with a delay time of 500 μs, and the corresponding linear fitting equations are (I-I0)/I0 = 14502·CGMP + 856 and (I-I0)/I0 = 2421·CRNA + 1926, respectively. The limits of detection to GMP and RNA are 0.021 μM and 0.71 ng/μL, respectively, and 1 can be used to determine the concentrations of GMP and RNA accurately. 1 displays rapid and stable luminescence response to the analytes, with the response times less than five seconds and no changes for the lanthanide emission over one week.

Luminescent molecularly imprinted polymer nanocomposites for emission intensity and lifetime rapid sensing of tenuazonic acid mycotoxin

Tenuazonic acid ((5S)-3-acetyl-5-[(2S)-butan-2-yl]-4-hydroxy-2,5-dihydro-1H-pyrrol-2-one, TeA) is a widespread Alternaria fungi mycotoxin in food produce. This toxin may be allergenic and provoke hay fever and asthma. Therefore, rapid methods to selectively detect TeA are needed. With this aim, we have engineered a novel trifunctional (red-luminescent, polymerizable, TeA-sensitive) ruthenium(II)-bipyridyl complex with 2,2′-biimidazole. Its peripheral N–H moieties recognize the enolate form of 1,3-dicarbonyl compounds (including TeA) in partially aqueous media. Such a binding decreases the luminescence intensity and lifetime (0.2 μs) of the Ru(II) probe. The probe also bears acrylate groups that allow radical copolymerization with methacrylamide and ethylene glycol dimethacrylate in the presence of TeA, to yield 9-nm thick luminescent molecularly imprinted polymer (MIP) shells onto 200-nm silica cores. The SiO2@Ru-MIP nanocomposite displays a very fast response (<5 s) suitable for real-time detection of TeA. No cross-sensitivity to other common food mycotoxins that also contain a β-hydroxycarbonyl moiety (alternariol, β-zeranol, cyclopiazonic acid) is observed. Detection limits of 63.8 μg L⁻1 and 75.2 μg L⁻1 under steady-state and time-resolved luminescence detection, respectively, have been measured without further optimization. The use of emission lifetime variations to monitor the levels of a target analyte has never been reported for a luminescent MIP-based sensor.

A miniaturized apparatus based on a smartphone for microsecond-resolved luminescence lifetime imaging

Time-resolved luminescence imaging is usually operated on Lab instruments equipped with high-speed excitation sources and detectors, which are always bulky and expensive. In order to miniaturize the instrument and reduce the cost, a FPGA (field programmable gate array) -based digital circuit was designed to control a mechanical chopper and a UV-LED for time-gated luminescence imaging. By utilizing a smartphone as a camera, the luminescence lifetime imaging of mouse kidney sections dyed by two europium complexes was successfully accomplished, demonstrating a temporal resolution of tens of microseconds. It is the first time that microsecond-resolved luminescence lifetime imaging was achieved with a smartphone. We believe this miniaturized apparatus can provide many opportunities for time-resolved luminescence detection and imaging for eliminating the background signals from scattering and short-lived autofluorescence.

Auto-Phase-Locked Time-Resolved Luminescence Detection: Principles, Applications, and Prospects.

Time-resolved luminescence measurement is a useful technique which can eliminate the background signals from scattering and short-lived autofluorescence. However, the relative instruments always require pulsed excitation sources and high-speed detectors. Moreover, the excitation and detecting shutter should be precisely synchronized by electronic phase matching circuitry, leading to expensiveness and high-complexity. To make time-resolved luminescence instruments simple and cheap, the automatic synchronization method was developed by using a mechanical chopper acted as both of the pulse generator and detection shutter. Therefore, the excitation and detection can be synchronized and locked automatically as the optical paths fixed. In this paper, we first introduced the time-resolved luminescence measurements and review the progress and current state of this field. Then, we discussed low-cost time-resolved techniques, especially chopper-based time-resolved luminescence detections. After that, we focused on auto-phase-locked method and some of its meaningful applications, such as time-gated luminescence imaging, spectrometer, and luminescence lifetime detection. Finally, we concluded with a brief outlook for auto-phase-locked time-resolved luminescence detection systems.

Time-resolved microfluidic flow cytometer for decoding luminescence lifetimes in the microsecond region.

Time-resolved luminescence detection using long-lived probes with lifetimes in the microsecond region have shown great potential in ultrasensitive and multiplexed bioanalysis. In flow cytometry, however, the long lifetime poses a significant challenge to measure wherein the detection window is often too short to determine the decay characteristics. Here we report a time-resolved microfluidic flow cytometer (tr-mFCM) incorporating an acoustic-focusing chip, which allows slowing down of the flow while providing the same detection conditions for every target, achieving accurate lifetime measurement free of autofluorescence interference. Through configuration of the flow velocity and detection aperture with respect to the time-gating sequence, a multi-cycle luminescence decay profile is captured for every event under maximum excitation and detection efficiency. A custom fitting algorithm is then developed to resolve europium-stained polymer microspheres as well as leukemia cells against abundant fluorescent particles, achieving counting efficiency approaching 100% and lifetime CVs (coefficient of variation) around 2-6%. We further demonstrate lifetime-multiplexed detection of prostate and bladder cancer cells stained with different europium probes. Our acoustic-focusing tr-mFCM offers a practical technique for rapid screening of biofluidic samples containing multiple cell types, especially in resource-limited environments such as regional and/or underdeveloped areas as well as for point-of-care applications.

Time-resolved luminescence detection of peroxynitrite using a reactivity-based lanthanide probe.

Peroxynitrite (ONOO−) is a powerful and short-lived oxidant formed in vivo, which can react with most biomolecules directly. To fully understand the roles of ONOO− in cell biology, improved methods for the selective detection and real-time analysis of ONOO− are needed. We present a water-soluble, luminescent europium(iii) probe for the rapid and sensitive detection of peroxynitrite in human serum, living cells and biological matrices. We have utilised the long luminescence lifetime of the probe to measure ONOO− in a time-resolved manner, effectively avoiding the influence of autofluorescence in biological samples. To demonstrate the utility of the Eu(iii) probe, we monitored the production of ONOO− in different cell lines, following treatment with a cold atmospheric plasma device commonly used in the clinic for skin wound treatment.

Enhanced Optics for Time-Resolved Singlet Oxygen Luminescence Detection

Singlet oxygen luminescence dosimetry (SOLD) is a highly promising direct monitoring method for photodynamic therapy (PDT) in the treatment of cancer. Early SOLD systems have been hampered by inefficient excitation, poor optical collection and immature infrared single photon detection technology. We report carefully engineered improvements addressing all of these deficiencies. We use a supercontinuum source with a tunable filter to precisely target the peak absorption wavelength of the chosen photosensitizer; we have designed a compact and versatile optical package for precise alignment; we have successfully employed state-of-the-art superconducting photon counting technologies. Through these improvements, we can achieve histogram acquisition from a photosensitizer in solution test sample. This setup opens the pathway to physiological SOLD studies for PDT dosimetry.

Smartphone-based apparatus for measuring upconversion luminescence lifetimes

Luminescence lifetime detection plays an important role in time-resolved detection and research. However, the traditional instruments always require expensive detectors such as time-correlated single photon counter or streak camera. Herein, a low-cost and miniaturized apparatus for measuring upconversion luminescence lifetimes was developed by using a smartphone equipped with a 980 nm CW laser and a motor. When the motor was driving the sample circling at a high linear velocity, the excited sample would emit a luminescence arc, which could be photographed by the phone camera. The rotating rate could be measured by a tuner APP and then used for transferring arc length to delay times. By analyzing the grayscale distribution of the luminescence arc, the luminescence decay curve was obtained, which was then used for exponential fit and calculating lifetimes. The images captured by different smartphones revealed similar lifetime values, suggesting a wide universality of this method. The whole system was not only remarkably cheaper but also more miniaturized than traditional instruments for measuring luminescence lifetimes, indicating the promising applications in point of care testing for time-resolved luminescence detection for bioanalysis and disease diagnosis.

Time- and spectrally resolved singlet oxygen phosphorescence detection—discriminating disturbance signals

Singlet oxygen is well known as the main mediator of photodynamic therapy. Therefore, great efforts are being made to detect singlet oxygen in many different biological environments. The most promising method is the time-resolved luminescence detection via its very weak near-infrared phosphorescence.However, in many biological environments these time-resolved measurements suffer from signal artifacts at short times (within the first 2 µs) after the excitation pulse. Neither the origin nor the kinetics of these disturbance signals have yet been successfully elucidated. Up to now, no way for eliminating them has been developed, be it mathematical or experimental.This work reports about a method for discrimination of the singlet oxygen phosphorescence from a disturbed signal by spectrally resolved measurements. We use a tiltable interference filter as an experimentally efficient and comparably cheap method to conduct highly sensitive time- and spectrally resolved luminescence measurements, inherently suitable for the future application in biological systems.We use Rose Bengal in water as an example system. It generates singlet oxygen at high quantum yield and shows additional luminescence emissions. The complex luminescence of Rose Bengal is treated as the disturbance signal. The isolation of the singlet oxygen phosphorescence by a fitting algorithm is demonstrated.

Singlet oxygen luminescence kinetics under PDI relevant conditions of pathogenic dermatophytes and molds

A treatment of onychomycosis using the photodynamic effect would be a favorable alternative to currently used antimycotic drugs. This study should be considered as a first step towards development and control of an efficient photodynamic inactivation of onychomycosis causative pathogens. Here, we evaluate the usage of time-resolved 2D singlet oxygen luminescence detection in combination with 2D fluorescence scanning as a tool to understand the behavior of the photosensitizer when applied to fungi on Petri dishes. To investigate the interaction of photosensitizer with fungi in various concentrations and in different stages of live, a photodynamic inactivation was avoided by keeping the samples in darkness. Scans of singlet oxygen luminescence and photosensitizer fluorescence were performed over a period of 24days. Two different photosensitizer, a cationic porphyrin and cationic corrole and two fungi strains, the dermatophyte Trichophyton rubrum and the mold Scopulariopsis brevicaulis, were investigated in this study. The two-dimensional correlation of photosensitizer fluorescence and singlet oxygen luminescence revealed differences in the diffusion of both photosensitizer. Even though the singlet oxygen luminescence was quenched with increasing growth of fungi, it was found that the kinetics of singlet oxygen luminescence could be detected on Petri dishes for both photosensitizers and both fungi strains for up to seven days.

Encapsulation-Stabilized, Europium Containing Nanoparticle as a Probe for Time-Resolved luminescence Detection of Cardiac Troponin I.

The use of a robust optical signaling probe with a high signal-to-noise ratio is important in the development of immunoassays. Lanthanide chelates are a promising material for this purpose, which provide time-resolved luminescence (TRL) due to their large Stokes shift and long luminescence lifetime. From this, they have attracted considerable interest in the in vitro diagnostics field. However, the direct use of lanthanide chelates is limited because their luminescent signal can be easily affected by various quenchers. To overcome this drawback, strategies that rely on the entrapment of lanthanide chelates inside nanoparticles, thereby enabling the protection of the lanthanide chelate from water, have been reported. However, the poor stability of the lanthanide-entrapped nanoparticles results in a significant fluctuation in TRL signal intensity, and this still remains a challenging issue. To address this, we have developed a Lanthanide chelate-Encapsulated Silica Nano Particle (LESNP) as a new immunosensing probe. In this approach, the lanthanide chelate is covalently crosslinked within the silane monomer during the silica nanoparticle formation. The resulting LESNP is physically stable and retains TRL properties of the parent lanthanide chelate. Using the probe, a highly sensitive, sandwich-based TRL immunoassay for the cardiac troponin I was conducted, exhibiting a limit of detection of 48 pg/mL. On the basis of the features of the LESNP such as TRL signaling capability, stability, and the ease of biofunctionalization, we expect that the LESNP can be widely applied in the development of TRL-based immunosensing.

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.

Prospects of in vivo singlet oxygen luminescence monitoring: Kinetics at different locations on living mice

BackgroundSinglet oxygen observation is considered a valuable tool to assess and optimize PDT treatment. In complex systems, such as tumors in vivo, only the direct, time-resolved singlet oxygen luminescence detection can give reliable information about generation and interaction of singlet oxygen. Up to now, evaluation of kinetics was not possible due to insufficient signal-to-noise ratio. Here we present high signal-to-noise ratio singlet oxygen luminescence kinetics obtained in mouse tumor model under PDT relevant conditions. MethodsA highly optimized system based on a custom made laser diode excitation source and a high aperture multi-furcated fiber, utilizing a photomultiplier tube with a multi photon counting device was used. ResultsLuminescence kinetics with unsurpassed signal-to-noise ratio were gained from tumor bearing nude mice in vivo upon topic application, subcutaneous injection as well as intravenous injection of different photosensitizers (chlorin e6 and dendrimer formulations of chlorin e6). Singlet oxygen kinetics in appropriate model systems are discussed to facilitate the interpretation of complex kinetics obtained from in vivo tumor tissue. ConclusionsThis is the first study addressing the complexity of singlet oxygen luminescence kinetics in tumor tissue. At present, further investigations are needed to fully explain the processes involved. Nevertheless, the high signal-to-noise ratio proves the applicability of direct time-resolved singlet oxygen luminescence detection as a prospective tool for monitoring photodynamic therapy.

Time-resolved singlet-oxygen luminescence detection with an efficient and practical semiconductor single-photon detector.

In clinical applications, such as PhotoDynamic Therapy, direct singlet-oxygen detection through its luminescence in the near-infrared range (1270 nm) has been a challenging task due to its low emission probability and the lack of suitable single-photon detectors. Here, we propose a practical setup based on a negative-feedback avalanche diode detector that is a viable alternative to the current state-of-the art for different clinical scenarios, especially where geometric collection efficiency is limited (e.g. fiber-based systems, confocal microscopy, scanning systems etc.). The proposed setup is characterized with Rose Bengal as a standard photosensitizer and it is used to measure the singlet-oxygen quantum yield of a new set of photosensitizers for site-selective photodynamic therapy.

Synthesis of a di(2-picolyl)amino-β-diketone dual-functional ligand that can coordinate to europium(III) for responding to copper(II) and sulfide ions

Abstract Lanthanide complex-based luminescent probes/chemosensors have shown great utilities in various biological and environmental assays with time-resolved detection mode to eliminate background noises. In this work, by conjugating di(2-picolyl)amine (DPA) with a tetradentate β -diketone 1,2-bis[4′-(1′′,1′′,1′′,2′′,2′′-pentafluoro-3′′,5′′-pentanedion-5′′-yl)benzyl]-4-chlorosulfo-benzene (BPPBCB), a novel dual-functional ligand that can coordinate to Eu 3+ for responding to Cu 2+ and S 2− ions in aqueous media, DPA–BPPBCB, has been designed and synthesized. The β -diketone moiety of DPA–BPPBCB can form a strongly luminescent complex with Eu 3+ . Upon reaction with Cu 2+ , accompanied by the formation of heterobimetallic complex Cu 2+ –DPA–BPPBCB–Eu 3+ , the Eu 3+ luminescence was quenched. While in the presence of S 2− , owing to the high affinity of S 2− to Cu 2+ , stable CuS was formed, which resulted in the release of Cu 2+ from Cu 2+ –DPA–BPPBCB–Eu 3+ , to restore the luminescence of the Eu 3+ complex. This unique “on-off-on” luminescence response of the Eu 3+ complex enabled Cu 2+ and S 2− ions in aqueous media to be detected with time-resolved luminescence detection mode.

Luminescence investigation of photosensitizer distribution in skin: correlation of singlet oxygen kinetics with the microarchitecture of the epidermis

This is the first study showing that singlet oxygen kinetics of topically applied photosensitizers coincides with the microarchitecture of skin, e.g., fissures and hair follicles. The kinetics indicate a chemical interaction of singlet oxygen with the skin, which allows differentiating between residual crème, e.g., in the follicular orifice, and photosensitizer penetrated into the skin. We show the feasibility of an easy-to-use fiber optic application providing the opportunity for in situ investigation, as well as a setup with focused optics for high-resolution two-dimensional scanning of singlet oxygen luminescence kinetics in skin samples. The results show that time-resolved singlet oxygen luminescence detection in tissue is a desirable tool for medical therapy, diagnostics, and evaluation of singlet oxygen interaction with biological environments.

Lexsyg — A new system for luminescence research

The newly developed lexsyg system by Freiberg Instruments is a versatile luminescence reader, suited for research on the luminescence of materials, fundamental research in luminescence dating, but also for routine mass measurements in retrospective dosimetry as well as in dating application. The 80 sample storage wheel is disconnected from the measurement chamber and therefore crosstalk of optical stimulation is absent and cross-irradiation is negligible from the α- and β-sources, which are providing very uniform irradiations, with the latter especially designed for radiofluores-cence (RF) measurement. Optical excitation sources and filter wheels to vary detection wavelengths can be programmed to change at almost any time within measurement sequences, including the auto-mated change of an optional wheel holding up to four different detectors. Thermoluminescence meas-urements and preheating are possible with a versatile heater, which can be programmed for linear or non-linear heating or cooling, as well as holding a temperature constant. Rates as well as durations can be varied, together with individual ramping, staging and cooling for an almost unlimited number of steps. Violet- and IR-lasers, green and blue LED-arrays can be operated in continuous (CW) or modulated mode (linear/non-linear), and optionally for pulsed as well as time resolved luminescence detection. Six arrays of power LEDs allow the simulation of different bleaching regimes (‘solar simu-lator’), while luminescence detection can be achieved by a variety of photomultiplier tubes and by CCD cameras for spatially resolved measurements and luminescence spectra.