Establishment of Double-antigen Sandwich Time-resolved Fluorescence Immunoassay for Detection of Pest des Petits Ruminants Virus

[Objectives]This study was conducted to explore rapid and large-scale screening and detection of peste des petits ruminants(PPR),so as to provide important technical means for prevention,control and purification of PPR.[Methods]Soluble N protein and NH fusion protein were successfully obtained in an Escherichia coli expression system by optimizing E.coli codon and expression conditions.Furthermore,based on purified soluble N protein and NH fusion protein,a double-antigen sandwich time-resolved fluorescence immunoassay method for detection of peste des petits ruminants virus(PPRV)was established.[Results]The method has high sensitivity and specificity and can specifically detect the antibody against PPRV in sheep serum,and it has no cross reaction with other related diseases.The method was used to detect 292 clinical samples,and compared with French IDVET competition ELISA kit.The coincidence rates of positive samples and negative samples from the two kinds of test kits were 92.47%and 97.26%,respectively,and the overall coincidence rate was 94.86%.The intra-group and inter-group coefficients of variation in the repeatability test were less than 10%.[Conclusions]Compared with the traditional ELISA method,the double-antigen sandwich time-resolved fluorescence immunoassay for detection of PPRV has equivalent sensitivity and specificity,and simple and rapid operation,and thus high application and popularization value.

Assembling Tunable Time-Resolved Fluorescence Layer onto Nano-Gold

The assembling of a coating of time-resolved fluorescent chelator BSPDA （ abbreviated for 4, 7-bis （ sulfhydrylphenyl）-1, 10-phenanthroline-2, 9-dicarboxylic acid） onto a nano-gold layer was demonstrated. First, BSPDA was synthesized by simple procedures, and then an approach was developed to immobilize BSPDA onto the nano-gold layer deposited on a silane modified glass substrate, whereby europium ion （Ⅲ, Eu^3＋ ） was captured and released owing to the interactive process of complexation and dissociation between BSPDA functionalized coating and Eu^3＋ solution. The fluorescence spectra and related lifetimes were determined. Also, the BSPDA functionalized coating＇s specific complexation with Eu^3＋ on the BSPDA assembly layer and the nonspecific adsorption of Eu^3＋ on the nano-gold layer were compared. These results allowed a selective complexation of Eu^3＋ by assembling a BSPDA chelating layer on the nano-gold layer; thus, a tunable time-resolved fluorescent layer was covalently attached, The results of the nanoparticle assembling and probing （or labeling） processes to specific bio-systems were very interesting and had significant implications to time-resolved-fluorescence-based detection on biosensor surfaces such as DNA chip and to arrayed light display devices.

Time-resolved Fluorescence Decay Study of Hematoporphyrin Derivative

Timefluorescence decays were first performed on hematoporphyrin derivative laser No.3 in buffer at different micelles concentration.The flu.orescence lifetime constants,measured with a time-correlated single photon counting system;could be fitted by two or three exponential components with different relative peak amplitudes depending on the observation wavelength^thus indicating the presence of different emitting molecular species and/or of molecular species in different aggregational states.

Time-Resolved Fluorescence Study of C_(60) Solution

Using a picosecond excitation source and a time-resolved streak camera detection system,the time-resolved fluorescence spectra of purified C_(60) have been recorded from toluene solution at room temperature.A fluorescence peak at about 730nm was detected and its lifetime was directly determined to be 1.1±0.1 ns.Two fluorescence peaks obtained from the mixture of C_(60) and C_(70) at 676 and 696nm were mainly related to C_(70) content.

Enhancement of rapid lifetime determination for time-resolved fluorescence imaging in forensic examination

An enhancement method of rapid lifetime determination is proposed for time-resolved fluorescence imaging to discriminate substances with approximate fluorescence lifetime in forensic examination. In the method, an image-exclusive-OR treatment with filter threshold adaptively chosen is presented to extract the region of interest from dual-gated fluorescence intensity images, and then the fluorescence lifetime image is reconstructed based on the rapid lifetime determination algorithm. Furthermore, a maximum and minimum threshold filtering is developed to automatically realize visualization enhancement of the lifetime image. In proof experiments, compared with traditional fluorescence intensity imaging and rapid lifetime determination method, the proposed method automatically distinguishes altered and obliterated documents written by two brands of highlighters with the same color and close fluorescence lifetime.

Communication Chain-foldability effect on coaggregation Evidence derived from time-resolved fluorescence spectroscopy

Very recently,we have proposed that all culprit molecules of arteriosclerosis mightpossess a common and measurable inherent property,namely,a coaggregating ten-dency.Extensive kinetic measurements of these tendencies in terms of △CAgC valueshave led to a new and surprising observation,i.e.,the cholesteryl ester with a longsaturated 18-carbon chain(CE-18)actually has a much smaller tendency towardcoaggregation than that of the ester with a“short”8-carbon chain(CE-8).Thisstate of affairs has been coined as the“chain-foldability effect”,i.e.,the 18-carbon

Energy transfer mechanism of PBS-thylakoid complexes——By time-resolved fluorescence spectra technique

IN a previous paper, we have studied the energy transfer mechanism among the PBS-thy-lakoid complex in detail by using steady-state spectra and deconvolution techniques. The ex-perimental results indicated that the energy transfer from PBS to two reaction centers of PS Ⅰand PS Ⅱ were parallel, and confirmed the model which was suggested by Mullineaxu.

TIME-RESOLVED FLUORESCENCE SPECTROSCOPY OF HEMATOPORPHYRIN DERIVATIVE LASER No. 3

Ⅰ. INTRODUCTIONPhotodynamic therapy (PDT) which utilizes hematoporphyrin derivative (HpD) as a photosensitizing drug has increasingly being used for the local treatment of malignant tumors. The success of this method is attributed to the ability of HpD to accumulate to

STEADY-STATE AND TIME-RESOLVED FLUORESCENCE STUDY OF TRIPLE EXCIPLEX IN THE SYSTEM OF POLYACENAPHTHALENE AND TEREPHTHALIC DIMETHYLESTER

Triple exciplex formed between polyacenaphthalene and terephthalic dimethylester (TDE) has been studied by means of the steady-state and time-resolved fluorescence spectra. The theoretical model of the triple exciplex formation for a flexible polymer chain in a dilute solution has been proposed. The fluorescence decays of the monomer, the exciplex and the triple exciplex obey a double exponential rule in the pbotophysical processes of the triple exciplex formation. The association rate constant of the exciplex formation is k_3=6.0×10~9s^(-1)(mol/L)^(-1); the association rate constant of the triple exciplex formation is k_6=5.2×10~7s^(-1). A mechanism for the triple exciplex formation from the exciplex to the triple exciplex has been proved through the noncrystal films corresponding to the same concentrations of the solution systems and the fluorescence lifetimes of the intramolecular excimer.

TIME-RESOLVED FLUORESCENCE STUDY OF TRIPLE EXCIPLEX FORMED IN THE SYSTEM OF POLY(2-VINYL) NAPHTHALENE AND TEREPHTHALIC DIMETHYLESTER

An intramolecular excimer of poly(2- vinyl) naphthalene was formed by non-adjacentchromophores interaction, then a triple exciplex was formed by interaction with the acceptormolecule further in dilute solution. The lifetime of the intramolecular excimer of poly(2-vinyl) naphthalene, τ_2 = 18.83 ns and the rate constant for the triple exciplex formation,k_7 = 4. 18× 10~9 (mol/L)^(-1)s^(-1), under diffusion-control were measured. An excimer or anexciplex could be an intermediate of the triple exciplex formation. A theoretical model ofthe triple exciplex formation is proposed.

NIR-II fluorescence imaging in liver tumor surgery: A narrative review

In liver tumor surgery,the recognition of tumor margin and radical resection of microcancer focis have always been the crucial points to reduce postoperative recurrence of tumor.However,naked-eye inspection and palpation have limited effectiveness in identifying tumor boundaries,and traditional imaging techniques cannot consistently locate tumors in real time.As an intraoperative real-time navigation imaging method,NIRfluorescence imaging has been extensively studied for its simplicity,reliable safety,and superior sensitivity,and is expected to improve the accuracy of liver tumor surgery.In recent years,the research focus of NIRfluorescence has gradually shifted from the-rst near-infrared window(NIR-I,700–900 nm)to the second near-infrared window(NIR-II,1000–1700 nm).Fluorescence imaging in NIR-II reduces the scattering effect of deep tissue,providing a preferable detection depth and spatial resolution while signi-cantly eliminating liver autofluorescence background to clarify tumor margin.Developingfluorophores combined with tumor antibodies will further improve the precision offluorescence-guided surgical navigation.With the development of a bunch offluorophores with phototherapy ability,NIR-II can integrate tumor detection and treatment to explore a new therapeutic strategy for liver cancer.Here,we review the recent progress of NIR-IIfluorescence technology in liver tumor surgery and discuss its challenges and potential development direction.

A novel fluorescence sensor for milk clotting enzyme chymosin using peptide as substrate and covalent organic framework nanosheet as fluorescence quencher

Chymosin is one of the critical enzymes in cheese making.Herein,we proposed a novel fluorometric assay for chymosin determination.Firstly,covalent organic frameworks(COF)were synthesized and exfoliated to 2-dimensional COF nanosheets(COF NS)by ultrasound treatment.Gold nanoparticles(Au NPs)were loaded with COF NS to prepare AuNPs/COF NS(Au@COF NS).Secondly,rhodamine B(RhB)modified substrate peptide(Pep)for chymosin was linked with Au@COF NS to construct a Pep-Au@COF NS nanocomposite.For the sensing principle,fluorescence of RhB was quenched by Au@COF NS and the fluorescence intensity was weak due to the fluorescence resonance energy transfer between COF NS and RhB of Pep.However,in the presence of chymosin,the RhB was released by specific cleavage of the substrate peptide by chymosin and resulted in the recovery of fluorescence.The increased fluorescence intensity was proportional to the increase of chymosin concentration and thus a“turn on”fluorescent sensor for chymosin was constructed.The sensor showed a linear range in the concentration of 0.05-60.00μg/mL for the detection of chymosin with a detection limit of 20 ng/mL.The sensor was used to quantify chymosin in rennet product with good selectivity,which has the potential applications in cheese manufacturing.

Ultrafast carrier dynamics in GeSn thin film based on time-resolved terahertz spectroscopy

We measure the time-resolved terahertz spectroscopy of GeSn thin film and studied the ultrafast dynamics of its photo-generated carriers.The experimental results show that there are photo-generated carriers in GeSn under femtosecond laser excitation at 2500 nm,and its pump-induced photoconductivity can be explained by the Drude–Smith model.The carrier recombination process is mainly dominated by defect-assisted Auger processes and defect capture.The firstand second-order recombination rates are obtained by the rate equation fitting,which are(2.6±1.1)×10^(-2)ps^(-1)and(6.6±1.8)×10^(-19)cm^(3)·ps^(-1),respectively.Meanwhile,we also obtain the diffusion length of photo-generated carriers in GeSn,which is about 0.4μm,and it changes with the pump delay time.These results are important for the GeSn-based infrared optoelectronic devices,and demonstrate that Ge Sn materials can be applied to high-speed optoelectronic detectors and other applications.

Time-resolved characteristics of a nanosecond pulsed multi-hollow needle plate packed bed dielectric barrier discharge

In this paper,self-designed multi-hollow needle electrodes are used as a high-voltage electrode in a packed bed dielectric barrier discharge reactor to facilitate fast gas flow through the active discharge area and achieve large-volume stable discharge.The dynamic characteristics of the plasma,the generated active species,and the energy transfer mechanisms in both positive discharge(PD)and negative discharge(ND)are investigated by using fast-exposure intensified charge coupled device(ICCD)images and time-resolved optical emission spectra.The experimental results show that the discharge intensity,number of discharge channels,and discharge volume are obviously enhanced when the multi-needle electrode is replaced by a multihollow needle electrode.During a single voltage pulse period,PD mainly develops in a streamer mode,which results in a stronger discharge current,luminous intensity,and E/N compared with the diffuse mode observed in ND.In PD,as the gap between dielectric beads changes from 0 to250μm,the discharge between the dielectric bead gap changes from a partial discharge to a standing filamentary micro-discharge,which allows the plasma to leave the local area and is conducive to the propagation of surface streamers.In ND,the discharge only appears as a diffusionlike mode between the gap of dielectric beads,regardless of whether there is a discharge gap.Moreover,the generation of excited states N_(2)^(+)(B^(2)∑_(u)^(+))and N2(C^(3)Π_(u))is mainly observed in PD,which is attributed to the higher E/N in PD than that in ND.However,the generation of the OH(A^(2)∑^(+))radical in ND is higher than in PD.It is not directly dominated by E/N,but mainly by the resonant energy transfer process between metastable N_(2)(A^(3)∑_(u)^(+))and OH(X^(2)Π).Furthermore,both PD and ND demonstrate obvious energy relaxation processes of electron-to-vibration and vibration-to-vibration,and no vibration-to-rotation energy relaxation process is observed.

Self-confocal NIR-II fluorescence microscopy for multifunctional in vivo imaging

Fluorescence imaging in the second near-infrared window(NIR-II,900–1880 nm)with less scattering background in biological tissues has been combined with the confocal microscopic system for achieving deep in vivo imaging with high spatial resolution.However,the traditional NIR-IIfluorescence confocal microscope with separate excitation focus and detection pinhole makes it possess low confocal e±ciency,as well as di±cultly to adjust.Two types of upgraded NIR-IIfluorescence confocal microscopes,sharing the same pinhole by excitation and emission focus,leading to higher confocal e±ciency,are built in this work.One type is-ber-pinhole-based confocal microscope applicable to CW laser excitation.It is constructed forfluorescence intensity imaging with large depth,high stabilization and low cost,which could replace multiphotonfluorescence microscopy in some applications(e.g.,cerebrovascular and hepatocellular imaging).The other type is air-pinhole-based confocal microscope applicable to femtosecond(fs)laser excitation.It can be employed not only for NIR-IIfluorescence intensity imaging,but also for multi-channelfluorescence lifetime imaging to recognize different structures with similarfluorescence spectrum.Moreover,it can be facilely combined with multiphotonfluorescence microscopy.A single fs pulsed laser is utilized to achieve up-conversion(visible multiphotonfluorescence)and down-conversion(NIR-II one-photonfluorescence)excitation simultaneously,extending imaging spectral channels,and thus facilitates multi-structure and multi-functional observation.

Research on a deconvolution algorithm for laser-induced fluorescence diagnosis based on the maximum entropy principle

Laser-induced fluorescence(LIF)spectroscopy is employed for plasma diagnosis,necessitating the utilization of deconvolution algorithms to isolate the Doppler effect from the raw spectral signal.However,direct deconvolution becomes invalid in the presence of noise as it leads to infinite amplification of high-frequency noise components.To address this issue,we propose a deconvolution algorithm based on the maximum entropy principle.We validate the effectiveness of the proposed algorithm by utilizing simulated LIF spectra at various noise levels(signal-to-noise ratio,SNR=20–80 d B)and measured LIF spectra with Xe as the working fluid.In the typical measured spectrum(SNR=26.23 d B)experiment,compared with the Gaussian filter and the Richardson–Lucy(R-L)algorithm,the proposed algorithm demonstrates an increase in SNR of 1.39 d B and 4.66 d B,respectively,along with a reduction in the root-meansquare error(RMSE)of 35%and 64%,respectively.Additionally,there is a decrease in the spectral angle(SA)of 0.05 and 0.11,respectively.In the high-quality spectrum(SNR=43.96 d B)experiment,the results show that the running time of the proposed algorithm is reduced by about98%compared with the R-L iterative algorithm.Moreover,the maximum entropy algorithm avoids parameter optimization settings and is more suitable for automatic implementation.In conclusion,the proposed algorithm can accurately resolve Doppler spectrum details while effectively suppressing noise,thus highlighting its advantage in LIF spectral deconvolution applications.

Near-Infrared Fluorescence Imaging Contrast Agents for Clinical Research: Limitations and Alternatives

Introduction: Near-infrared fluorescence imaging is a technique that will establish itself in the short term at the international level because it is recognized for its potential to improve the performance of surgical interventions, its moderate investment and operating costs and its portability. Although the technology is now mature, there is currently the problem of the availability of contrast agents to be injected IV. The aim of this methodology article is to propose an alternative solution to the need for contrast agents for clinical research, particularly in oncology. Methodology: They consist of coupling a fluorescent marker in the form of an NHS derivative, such as IR DYE manufactured in compliance with GMP, with therapeutic monoclonal antibodies having marketing authorization for molecular imaging. For a given antibody, the marking procedure must be the subject of a validation file on the final preparation filtered on a sterilizing membrane at 0.22 μm. Once the procedure has been validated, it would be unnecessary to repeat the tests before each clinical research examination. A check of the marking by thin-layer chromatography (TLC) and place it in a sample bank at +4˚C for 1 month of each injected formulation would be sufficient for additional tests if necessary. Conclusion: Molecular near-infrared fluorescence imaging is experiencing development, the process of which could be accelerated by greater availability of clinical contrast agents. Alternative solutions are therefore necessary to promote clinical research in this area. These methods must be shared to make it easier for researchers.

Isomeric fluorescence sensors for wide range detection of ionizing radiations

In order to achieve a wider range of ionizing radiations detection,novel fluorescence sensing materials have been developed that utilize the fluorescence enhancement phenomenon caused by the intramolecular photoinduced electron transfer(PET)effect.Two perylene diimide isomers PDI-P and PDI-B were designed and synthesized,and their molecular structures were characterized by high-resolution Fourier transform mass spectrometry(HRMS),nuclear magnetic resonance hydrogen and carbon spectroscopy(~1H and~(13)C NMR).The interaction between ionizing radiation and fluorescent molecules was simulated by HCl titration.The results show that combining PDIs and HCl can improve fluorescence through the retro-PET process.Despite the similarities in chemical structures,the fluorescent enhancement multiple of PDI-B with aromatic amine as electron donor is much higher than that of PDI-P with alkyl amine.In the direct irradiation experiments of ionizing radiation,the emission enhancement multiples of PDI-P and PDI-B are 2.01 and 45.4,respectively.Furthermore,density functional theory(DFT)and time-dependent density functional theory(TDDFT)calculations indicate that the HOMO and HOMO-1 energy ranges of PDI-P and PDI-B are 0.54 e V and 1.13 e V,respectively.A wider energy range has a stronger driving force on electrons,which is conducive to fluorescence quenching.Both femtosecond transient absorption spectroscopy(fs-TAS)and transient fluorescence spectroscopy(TFS)tests show that PDI-B has shorter charge separation lifetime and higher electron transfer rate constant.Although both isomers can significantly reduce LOD during PET process,PDI-B with aromatic amine has a wider detection range of 0.118—240 Gy due to its larger emission enhancement,which is a leap of three orders of magnitude.It breaks through the detection range of gamma radiation reported in existing studies,and provides theoretical support for the further study of sensitive and effective new materials for ionizing radiation detection.

In vivo fluorescence flow cytometry reveals that the nanoparticle tumor vaccine OVA@HA-PEI effectively clears circulating tumor cells

Tumor vaccine therapy offers significant advantages over conventional treatments,including reduced toxic side effects.However,it currently functions primarily as an adjuvant treatment modality in clinical oncology due to limitations in tumor antigen selection and delivery methods.Tumor vaccines often fail to elicit a sufficiently robust immune response against progressive tumors,thereby limiting their clinical efficacy.In this study,we developed a nanoparticle-based tumor vaccine,OVA@HA-PEI,utilizing ovalbumin(OVA)as the presenting antigen and hyaluronic acid(HA)and polyethyleneimine(PEI)as adjuvants and carriers.This formulation significantly enhanced the proliferation of immune cells and cytokines,such as CD3,CD8,interferon-,and tumor necrosis factor-,in vivo,effectively activating an immune response against B16–F10 tumors.In vivofluorescenceflow cytometry(IVFC)has already become an effective method for monitoring circulating tumor cells(CTCs)due to its direct,noninvasive,and long-term detection capabilities.Our study utilized a laboratory-constructed IVFC system to monitor the immune processes induced by the OVA@HA-PEI tumor vaccine and an anti-programmed death-1(PD-1)antibody.The results demonstrated that the combined treatment of OVA@HA-PEI and anti-PD-1 antibody significantly improved the survival time of mice compared to anti-PD-1 antibody treatment alone.Additionally,this combination therapy substantially reduced the number of CTCs in vivo,increased the clearance rate of CTCs by the immune system,and slowed tumor progression.Thesefindings greatly enhance the clinical application prospects of IVFC and tumor vaccines.

Pay attention to the application of indocyanine green fluorescence imaging technology in laparoscopic liver cancer resection

Traditional laparoscopic liver cancer resection faces challenges,such as difficultiesin tumor localization and accurate marking of liver segments,as well as theinability to provide real-time intraoperative navigation.This approach falls shortof meeting the demands for precise and anatomical liver resection.The introductionof fluorescence imaging technology,particularly indocyanine green,hasdemonstrated significant advantages in visualizing bile ducts,tumor localization,segment staining,microscopic lesion display,margin examination,and lymphnode visualization.This technology addresses the inherent limitations oftraditional laparoscopy,which lacks direct tactile feedback,and is increasinglybecoming the standard in laparoscopic procedures.Guided by fluorescenceimaging technology,laparoscopic liver cancer resection is poised to become thepredominant technique for liver tumor removal,enhancing the accuracy,safetyand efficiency of the procedure.