Synthesis and Spectral Properties of Novel Water-soluble Near-infrared Fluorescent Indocyanines

Two fluorescent pentamethine and a squarylium indocyanines containing at least one p-carboxybenzyl group on N atoms in the heterocyclic rings were synthesized. They had good water solubility and photostability. Their maximum absorption and maximum emission were 600-700 nm in water. When it was anchored onto nanostructured TiO2 electrode, compared with in water, the squaraine showed double absorption peaks (one blue shifted and another red shifted) and absorption intensity of the red shift peak increased with the increase of the time of irradiation. The intensity of the blue one decreased simultaneously. We proposed that the presence of two electronic charge forms of squaraine anchored on the TiO2 film might be the reason.

A New Method for Ultra-sensitive P24 Antigen Assay Based on Near-infrared Fluorescent Microsphere Immunochromatography

Objective To develop a rapid,highly sensitive quantitative method for detecting P24 antigen based on near-infrared fluorescent microsphere immunochromatography.Methods First,we prepared a lateral flow assay test strip,and labeled the detection antibody using a fluorescent microsphere.Second,we optimized the antibody labeling conditions.Third,we optimized the detection conditions.Fourth,we created a working curve.Fifth,we conducted a methodological assessment of the established fluorescent microsphere immunochromatography method.Sixty-six clinical samples were tested,and we compared the established fluorescent microsphere immunochromatography with the quantitative ELISA method.Results According to the working curve,the detection limit of the method is 3.4 pg/mL,and the detection range is 3.4 pg/mL to 10 ng/mL.The average intra-assay recovery was 99.6%,and the Coefficient of Variation(CV)was 5.4%–8.6%;the average inter-assay recovery was 97.3%,and the CV was 8.5%–11%.The detection rate of fluorescent microsphere immunochromatography was higher than ELISA method,and had a good correlation with ELISA.Conclusion The P24 antigen quantitative detection method based on near-infrared fluorescent microsphere immunochromatography has the advantages of rapid detection,high sensitivity,and wide detection range;thus,it is suitable for early clinical diagnosis and continuous monitoring of AIDS.

Synthesis of New Near-infrared Fluorescent Dyes

A new near-infrared fluorescent dye, 9-N-(2-hydroxyethyl)-N-methylamino-6-carbethoxy-5H-benzo[a]phenoxazin-5-one 1, was prepared from the reaction of N-(2-hydroxyethyl)-N-methyl-4-nitrosoaniline hydrochloride and ethyl 1,3-dihydroxynaphthoate. Five more fluorescent compounds were synthesized by the reaction of the resulting dye 1 with appropriate amino acid or carboxylic acids.

A NEAR-INFRARED FLUORESCENT DEOXYGLUCOSE DERIVATIVE FOR OPTICAL IMAGING OF EXPERIMENTAL ARTHRITIS

The purpose of this study is to investigate whether a near-infrared fluorescence(NIRF)probe,Cy5.5-d-glucosamine(Cy5.5-2DG),can image arthritis in collagen-induced arthritic(CIA)mice.The presence of arthritis was verified by both visual examination and micro-computed tomography(MicroCT)imaging.CIA mice were imaged by a micro-positron emission tomography(MicroPET)scanner one hour after intravenous injection of 2-deoxy-2-[18F]fluoro-d-glucose([18F]FDG).After radioactivity of[18F]FDG decayed away,Cy5.5-2DG was injected into a lateral tail vein of the mice.Arthritic tissue targeting and retention of Cy5.5-2DG in CIA mice were evaluated and quantified by an optical imaging system.Inflammatory tissue in CIA mice was clearly visualized by[18F]FDG-MicroPET scan.NIRF imaging of Cy5.5-2DG in the same mice revealed that the pattern of localization of Cy5.5-2DG in the arthritic tissue was very similar to that of[18F]FDG.Quantification analysis further showed that[18F]FDG uptake in arthritic tissues at one hour post-injection(p.i.)and Cy5.5-2DG uptakes at different time points p.i.were all well correlated(r2 over 0.65).In conclusion,Cy5.5-DG can detect arthritic tissues in living mice.The good correlation between the[18F]FDG uptake and Cy5.5-2DG accumulation in the same arthritic tissue warrants further investigation of Cy5.5-2DG as an approach for assessment of anti-inflammatory treatments.

Near-infrared fluorescent labeled CGRRAGGSC peptides for optical imaging of IL-11Rα in athymic mice bearing tumor xenografts

The interleukin-11(IL-11)and the IL-11 receptorα-subunit(IL-11Rα)have been demonstrated to regulate the invasion and proliferation of tumor cells.Our study intends to evaluate a noninvasive imaging of IL-11Rαexpression in breast tumors using near-infrared(NIR)fluorescent dye Cy7-labeled IL-11 mimic peptide CGRRAGGSC.This work evaluated the IL-11Rαexpression of breast tumor cells and the binding status of this peptide to IL-11Rαin vitro and in vivo by using Western blotting,immunofluorescence staining and near-infrared fluorescence imaging.Our biochemical study showed that IL-11Rαwas overexpressed in breast tumor cells(MCF-7).The cell-binding assay demonstrated specific binding of peptide CGRRAGGSC to MCF-7 cells in vitro.In vivo imaging results showed that NIR fluorescent signals of Cy7-CGRRAGGSC were selectively accumulated in tumor and metabolic organs.While in the blocking experiment,free CGRRAGGSC obviously blocked the concentration of the Cy7-CGRRAGGSC in the tumors.These results suggested that IL-11Rαmay be used as a potential target for noninvasive imaging in IL-11Rαoverexpressed tumors.Furthermore,the imaging agent of near-infrared fluorescent dye Cy7-labeled CGRRAGGSC is suitable for IL-11Rαexpression imaging study in vivo.

A New Heptamethine Cyanine-Based Near-Infrared Fluorescent Probe for Divalent Copper Ions with High Selectivity

A new near-infrared fluorophore 2-(2-Aminoethyl) pyridine-tricarbocyanine (1) was rationally designed and synthe-sized as a fluorescent probe for detection of Cu2+ with high selectivity. The response of Probe 1 is based on the fluorescence quenching upon binding to Cu2+. The sensing performance of the proposed Cu2+-sensitive Probe 1 was then investigated. The probe can be applied to the quantification detection of Cu2+ with a linear concentration range covering from 4.8 × 10-7 to 1.6 × 10-4 mol/L and a detection limit of 9.3 × 10-8 mol/L. The experimental results showed that the response of 1 to Cu2+ was independent of pH in medium condition (pH 6.0-8.0), and exhibited excellent selectivity towards Cu2+ over other common metal cations.

A ratiometric near-infrared fluorescent probe for the detection and monitoring of hypochlorous acid in rheumatoid arthritis model and real water samples

Recent studies revealed that the increased level of hypochlorous acid(HOCl)may be deemed to be one of the signs of chronic inflammatory joint disease.Accordingly,the development of effective methods for rapid and accurate detection or monitoring of HOCl in vivo is of great significance for further understanding the role of HOCl in rheumatoid arthritis(RA).Herein,a ratiometric near-infrared(NIR)fluorescent probe(PTA)was reported for the detection and monitoring of HOCl.In the presence of HOCl,the electron-rich S atom and C=C double bond of probe PTA were oxidized in sequence,resulting in the significant hypochromatic shift and decline of absorption spectra.Simultaneously,the intramolecular charge transfer(ICT)process of PTA is inhibited,causing the intrinsic fluorescence emission of PTA shift from 680 to 550 nm.PTA-based test paper strips were successfully prepared and applied to determinate HOCl in actual water samples by“naked eye”colorimetric method.PTA features NIR emission,large Stokes shift(200 nm),low cytotoxicity,high sensitivity(33.9 nM),and short response time(45 s),which enable it to be successfully utilized for imaging endogenous and exogenous HOCl in living zebrafish and mice.More importantly,PTA shows remarkable effectiveness for the monitoring of HOCl-mediated treatment response to RA.Consequently,PTA provides a new approach to further understand the role of HOCl in RA and evaluate the drug treatment efficiency of RA.

A near-infrared fluorescent probe for visualizing transformation pathway of Cys/Hcy and H2S and its applications in living system

Sulfydryl-contained(-SH)substances including hydrogen sulfide(H_(2)S),cysteine(Cys),homocysteine(Hcy)and glutathione(GSH)play crucial roles in living systems,and their variations are closely associated with various diseases.Herein,we developed a near-infrared intramolecular charge transfer(ICT)based fluorescent probe Y-NBD,achieving detection of Cys/Hcy and H_(2)S with different fluorescent signals(green-red for Cys/Hcy,red for H_(2)S),large Stokes shifts(∼100/105nm or 191 nm)and high signal-background-ratio,but not responding to GSH.Y-NBD was successfully applied to image exogenous/endogenous Cys/Hcy and H_(2)S in various living cancer cells(HeLa,A549,and HepG2)and in zebrafish.It not only visualized the transformation pathway of several thiols in HepG2 cells but also verified that the intestine is the main site for the activation and metabolism of Y-NBD in zebrafish,as well as realized to evaluate the degree of drug-induced liver injury.This work provides a promising tool for imaging Cys/Hcy and H_(2)S in living systems and shows great potency in evaluating drug-induced liver injury and its treatment.

A near-infrared fluorescent probe for fast and precise imaging of senescent cells and ovarian cancer cells via trackingβ-galactosidase

Aging-related diseases are gradually becoming a major problem with the rapid development of aged population in human society.Although many fluorescent probes have been employed to diagnosis senescence via imaging senescence-associatedβ-galactosidase(SA-β-Gal),which is proved to be closely associated with senescent cells,the similar catalytic effectiveness of enzymatic reaction of ovarian cancer-associatedβ-Gal(OA-β-Gal)will interfere with imaging accuracy.Herein,a near-infrared(NIR)hemicyanine based fluorescent probe HCyXA-βGal was designed for light-up imaging of live cells containingβ-Gal.With the organelle-targeting morpholinyl and positive charge moieties,HCyxA-βGal was successfully applicated to image the difference of enzymatic location in senescent cells and ovarian cancer cells.Furthermore,inspired by the fast response performance,fast and precise imaging of the two cell lines was realized via covering another dimension of fluorescence signal:time-dependent intensity.

Near-infrared fluorescent probes for imaging of amyloid plaques in Alzheimer's disease

One of the early pathological hallmarks of Alzheimer's disease(AD) is the deposition of amyloid-β(Aβ) plaques in the brain. There has been a tremendous interest in the development of Aβ plaques imaging probes for early diagnosis of AD in the past decades. Optical imaging, particularly near-infrared fluorescence(NIRF) imaging, has emerged as a safe, low cost, real-time, and widely available technique, providing an attractive approach for in vivo detection of Aβ plaques among many different imaging techniques. In this review,we provide a brief overview of the state-of-the-art development of NIRF Aβ probes and their in vitro and in vivo applications with special focus on design strategies and optical, binding, and brain-kinetic properties.

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.

Emerging applications of near-infrared fluorescent metal nanoclusters for biological imaging

Fluorescent metal nanoclusters（MNCs） have recently emerged as a novel kind of promising fluorescent probes for biological imaging because of their ultrasmall core size（〈2 nm）, strong photoluminescence,facile availability and good biocompatibility. In this review, we provide an update on recent advances in the development of near infrared（NIR）-emitting MNCs in terms of synthesis strategies and bioimaging applications. We mainly focus on the utilization of NIR-emitting MNCs（including Au, Ag, Cu and alloy NCs） either as single modal imaging（fluorescence intensity-based imaging, fluorescence lifetime imaging, two-photon imaging） probes or as multimodal imaging（such as NIR fluorescence/X-ray computed tomography/magnetic resonance imaging, NIR fluorescence/photoacoustic imaging/magnetic resonance imaging, NIR fluorescence/single photon emission computed tomography） probes in biological cells and tissues. Finally, we give a brief outlook on the future challenges and prospects of developing NIR-emitting MNCs for bioimaging.

A near-infrared fluorescent probe for monitoring fluvastatin-stimulated endogenous H_2S production

Most reported fluorescent probes have limitations in practical applications in living systems due to the strong autofluorescence background,construction of probes with near-infrared（NIR） fluorescence emission is an accessible approach for addressing this challenge.We here designed a NIR fluorescent probe for monitoring the endogenous production of H2S in living cells.The designed probe showed significant NIR fluorescence turn-on response to H2S with high selectivity,enabling the sensitive detection H2S.Importantly,the probe could be applied in monitoring the endogenous production of H2S in raw 264.7 macrophages.This study showed that fluvastatin can promote the activity of cystathionineγ-lyase（CSE） for generation H2S.

Molecularly near-infrared fluorescent theranostics for in vivo tracking tumor-specific chemotherapy

Molecularly near-infrared(NIR) theranostics, combining in vivo sensing and tumor-specific therapeutic capability within one molecular system, have received considerable attention in recent years. Compared with the visible fluorescence imaging, NIR imaging(emission wavelength at 650–900 nm) possesses unique advantages including the minimum photodamage to biological samples, deep penetration, and low interference from auto-fluorescence. In over past decades, there has been an explosive development in the design of molecular imaging contrasts and imaging-guided therapeutics. In this review, we have sumarried the strategies of the NIR theranostics for imaging and tumor-specific chemotherapy applications in living systems. It is noted that the molecularly NIR theranostic design strategy could address current challenges of real-time in vivo sense-and-release for the intelligent biosensing and personalized treatment.

A pinacol boronate caged NIAD-4 derivative as a near-infrared fluorescent probe for fast and selective detection of hypochlorous acid

Hypochlorous acid （HOCI） is one of highly reactive oxygen species （ROS）. It is involved in both immune defense against invading microbes and the progression of many diseases including cardiovascular disease and neurodegeneration disorders. It is generated from hydrogen peroxide （H2O2） and chloride ions in the presence of myeloperoxidase in activated neutrophils. To illustrate HOCI＇s biological functions, fluorescent probes, particularly those fluorescence emissions are in the near-infrared range, are highly needed for in vivo applications. Herein, we reported the design of a pinacol boronate caged near-infrared （NIR） fluorescent probe I derived from an Aft binding fluorophore NIAD-4 for fast and selective detection of HOC1/C10 over other ROS. Upon exposure to HOCI/CIO-, the pinacol boronate caging group of the probe 1 was quickly converted to electron-donating hydroxyl group, which increased intramolecular charge transfer OCT） in the excited state and resulted in the red-shift and intensity enhancement of fluorescence emission. The probe bears several unique features： （1） It could be used as either a ratiomatic or turn-on fluorescent probe; （2） Reaction of the caging group boronate with HOCI is very fast and finishes within seconds, which provides the selectivity over H2O2; （3） The NIAD-4 fluorophore provides additional selectivity for detection of HOCl over peroxynitrite. Moreover, the utility of the probe in imaging HOCllClO- was demonstrated in in vitro phantom imaging studies using mouse brain homogenate as biological relevant media.

An activatable ratiometric near-infrared fluorescent probe for hydrogen sulfide imaging in vivo

Ratiometric fluorescent probes hold great promise for in vivo imaging;however,stimuli-activatable ratiometric probes with fluorescence emissions in near-infrared(NIR)region are still very few.Herein,we report a hydrogen sulfide(H_2S)-activatable ratiometric NIR fluorescent probe(1-SPN)by integrating a H_2S-responsive NIR fluorescent probe 1 into a H_2S-inert poly[2,6-(4,4-bis-(2-ethylhexyl)-4 H-cyclopenta[2,1-b;3,4-b′]dithiophene)-alt-4,7(2,1,3-benzothiadiazole)](PCPDTBT)-based NIR semiconducting polymer nanoparticle(SPN).1-SPN shows"always on"PCPDTBT fluorescence at 830 nm and weak probe 1 fluorescence at 725 nm under excitation at 680 nm.The ratio of NIR fluorescence intensities between 725 and 830 nm(I_(725)/I_(830))is small.Upon interaction with H_2S,the fluorescence at 725 nm is rapidly switched on,resulting in a large enhancement of I_(725)/I_(830),which is allowed for sensitive visualization and quantification of H_2S concentrations in living cells.Taking advantage of enhanced tissue penetration depth of NIR fluorescence,1-SPN is also applied for real-time ratiometric fluorescence imaging of hepatic and tumor H_2S in living mice.This study demonstrates that activatable ratiometric NIR fluorescent probes hold great potential for in vivo imaging.

A practical strategy to develop isoform-selective near-infrared fluorescent probes for human cytochrome P450 enzymes

Currently,the development of selective fluorescent probes toward targeted enzymes is still a great challenge,due to the existence of numerous isoenzymes that share similar catalytic capacity.Herein,a double-filtering strategy was established to effectively develop isoenzyme-specific fluorescent probe(s)for cytochrome P450(CYP)which are key enzymes involving in metabolism of endogenous substances and drugs.In the first-stage of our filtering approach,near-infrared(NIR)fluorophores with alkoxyl group were prepared for the screening of CYP-activated fluorescent substrates using a CYPs-dependent incubation system.In the second stage of our filtering approach,these candidates were further screened using reverse protein-ligand docking to effectively determine CYP isoenzyme-specific probe(s).Using our double-filtering approach,probes S9 and S10 were successfully developed for the real-time and selective detection of CYP2C9 and CYP2J2,respectively,to facilitate high-throughput screening and assessment of CYP2C9-mediated clinical drug interaction risks and CYP2J2-associated disease diagnosis.These observations suggest that our strategy could be used to develop the isoform-specific probes for CYPs.

Effects of the molluscicide can didate PPU06 on alkaline phosphatase in the golden apple snails determined using a near-infrared fluorescent probe

We constructed a reaction-based near-infrared fluorescent probe(Niap) to specifically identify alkaline phosphatase(ALP) with fast red fluorescence enhancement.Based on the positive concentrationdependent manner between the fluorescent intensity of the Niap and ALP,probe Niap was used to study the ALP enrichment and variation in golden apple snails(Pomacea canaliculata) exposed to the molluscicide candidate PPU06.After treatment with different concentrations of PPU06 over various times,three organs of the surviving snails,liver,stomach and plantaris,were frozen and sectioned for fluorescent imaging experiments.With increased PPU06 concentration,red fluorescence substantially increased in the liver and reached a maximum within 24 h when the PPU06 co ncentration was 0.75 mg/L.No obvious changes in the stomach or foot plantaris were found.It showed PPU06 caused liver injury and stimulated the increase of ALP in the liver of P.canaliculata.This study demonstrates a rapid ALP fluorescent identification method that can be used to study the effects of PPU06 on P.canaliculata.It also provides optical evidence that may aid in the discovery of new chemistry for snail control.

A near-infrared fluorescent probe for fluorine ions and its application in the imaging of HepG2 cells

In this study, we have designed and synthesized a new near-infrared (NIR) fluorescent probe (BODIPY-Se) to detect fluorine ions (F-) using a B-Se bond to connect the fluorescent dye BODIPY and benzotrifluoride. The probe exhibited a highly selective fluorescence response to F- with a detection limit of 7.4×108 mol/L. The excitation and emission spectra of the probe in the NIR region avoid background fluorescence interference present in biological systems. The fluorescent imaging of HepG2 cells demonstrated that the newly developed probe should be broadly applicable to detect F- in living cells.

A Near-infrared Fluorescent Probe for Selective Simultaneous Detection of Fe2＋ and CI- in Living Cells

A naked-eyed chromo-near infrared fluorescent probe for simultaneous detection of Fe2＋ and C1 has been developed, based on photoinduced electron transfer mechanism （PET）. The fluorophore is cyanine （Cy-SO3）, and the Fe2＋ receptor is 4＇-（aminomethylphenyl）-2,2＇,6＇,2＂-terpyridine （Tpy）. The probe responds linearly and rapidly to [Fe2＋] and [C1 ] variations under physiological conditions and exhibits high sensitivity, good photostability, and excellent cell membrane permeability. The real-time imaging of cellular Fe2＋ was achieved successfully in living HL-7702, HepG2, and RAW264.7 cell lines.