Specific Fluorescence Research Articles

Development of Specific and Sensitive Fluorescence Probes for Glucagon Utilizing Peptides from Receptor’s Recognition Sites

Development of Specific and Sensitive Fluorescence Probes for Glucagon Utilizing Peptides from Receptor’s Recognition Sites

Precise Electron-Withdrawing Strength Modulation of ESIPT Probes for Ultrasensitive and Specific Fluorescence Sensing.

The precise regulation of the electron-withdrawing/electron-donating strength in a probe is of great significance for the design of reaction-based fluorescent probes with specific functionalities. Here, a family of excited-state intramolecular proton transfer (ESIPT)-based probes with fluorescence turn-on sensing properties toward KMnO4 was designed by precisely modulating the electron-withdrawing strength of the substituents located at the para-position of the recognition group. It is found that -F, -CHO, and -H as the electron-withdrawing groups bound at the probe can specifically recognize KMnO4, which ensures a blue emission displayed by the reaction products. Especially with -CHO as the electron-withdrawing group, the reaction product shows the most stable fluorescence. The probe 2-(benzo[d]oxazol-2-yl)-4-formylphenyl acrylate (BOPA-CHO) demonstrated a more superior sensing performance toward KMnO4, including a low limit of detection (LOD, 0.96 nM), a rapid response (<3 s), and a rather good selectivity even in the presence of 21 interferents. Moreover, the practicality of the probe was further verified by a test pen comprising a BOPA-CHO-embedded sponge, which is capable of detecting KMnO4 solid with a naked-eye LOD of 11.62 ng. The present probe design and modulation strategy would open up a new path for the design of high-performance fluorescent probes.

Zebrafish thrombosis models according to the location of thrombus formation.

Ischemic stroke becomes a major cause of death and disability. It can develop due to intravascular or cardiac thromboemboli. Animal models that reflect diverse stroke mechanisms remain under development. Using photochemical thrombosis, we developed a feasible zebrafish model according to the thrombus location (intracerebral vs. intracardiac). We validated the model using real-time imaging and thrombolytic agent. We used transgenic zebrafish larvae (flk:gfp), which express specific fluorescence in endothelial cells. We injected Rose Bengal, a photosensitizer as a mixture of photosensitizer, and a fluorescent agent into the cardinal vein of the larvae. We then evaluated real-time thrombosis in vivo by inducing thrombosis through exposure to a confocal laser (560 nm) and staining the blood flow (RITC-dextran). We validated intracerebral and intracardiac thrombotic models with checking the activity of tissue plasminogen activator (tPA). The photochemical agent induced the formation of intracerebral thrombi in transgenic zebrafish. Real-time imaging techniques confirmed the formation of the thrombi. The damage and apoptosis of the vessel's endothelial cells were seen in the in vivo model. An intracardiac thrombosis model was developed by the same method using photothrombosis, and the model was validated through thrombolysis by tPA. We developed and validated two zebrafish thrombosis models that are readily available, cost-effective, and intuitive for assessing the efficacy of thrombolytic agents. These models can be used for a broad spectrum of future studies, such as screening and efficacy assessment of new antithrombotic agents.

General Strategy for Specific Fluorescence Imaging of Homocysteine in Living Cells and In Vivo.

The aberrantly changed level of homocysteine (Hcy) triggers a variety of pathological symptoms and subsequently Hcy-related diseases. Direct and selective visualization of Hcy in biological systems is pivotal to understanding the pathological functions of Hcy at the molecular level. Herein, a general strategy was developed for the specific fluorescence imaging of Hcy through the combination of dual-binding sites and the introduction of a nitro group at the 6-position of the 7-diethylaminocoumarin fluorophore. Also, a series of novel fluorescent probes were exploited for monitoring Hcy with excellent selectivity, high sensitivity, and far-red/near-infrared fluorescence emission. Furthermore, fluorescence imaging of endogenous Hcy dynamics in living cells and in vivo was achieved, providing direct and solid evidence for the increasement of endogenous Hcy in type 2 diabetes mellitus and Alzheimer's disease. This research will greatly advance the development and understanding of the molecular nexus between the Hcy metabolism cascade and the root causes of diseases related to Hcy.

Blocking autofluorescence in brain tissues affected by ischemic stroke, hemorrhagic stroke, or traumatic brain injury.

Autofluorescence is frequently observed in animal tissues, interfering with an experimental analysis and leading to inaccurate results. Sudan black B (SBB) is a staining dye widely used in histological studies to eliminate autofluorescence. In this study, our objective was to characterize brain tissue autofluorescence present in three models of acute brain injury, including collagenase-induced intracerebral hemorrhage (ICH), traumatic brain injury (TBI), and middle cerebral artery occlusion, and to establish a simple method to block autofluorescence effectively. Using fluorescence microscopy, we examined autofluorescence in brain sections affected by ICH and TBI. In addition, we optimized a protocol to block autofluorescence with SBB pretreatment and evaluated the reduction in fluorescence intensity. Compared to untreated, pretreatment with SBB reduced brain tissue autofluorescence in the ICH model by 73.68% (FITC), 76.05% (Tx Red), and 71.88% (DAPI), respectively. In the TBI model, the ratio of pretreatment to untreated decreased by 56.85% (FITC), 44.28% (Tx Red), and 46.36% (DAPI), respectively. Furthermore, we tested the applicability of the protocol using immunofluorescence staining or Cyanine-5.5 labeling in the three models. SBB treatment is highly effective and can be applied to immunofluorescence and fluorescence label imaging techniques. SBB pretreatment effectively reduced background fluorescence but did not significantly reduce the specific fluorescence signal and greatly improved the signal-to-noise ratio of fluorescence imaging. In conclusion, the optimized SBB pretreatment protocol blocks brain section autofluorescence of the three acute brain injury models.

Enhancing the Activity of a Self-Inducible Promoter in Escherichia coli through Saturation Mutation and High-Throughput Screening

The use of self-inducible promoters is a promising strategy to address metabolic imbalances caused by overexpression. However, the low activity of natural self-inducible promoters hinders their widespread application. To overcome this limitation, we selected the fic promoter as a model promoter to create an enhanced self-inducible promoter library using saturation mutations and high-throughput screening. Sequence analysis revealed that these promoters share certain characteristics, including semi-conservation in the −35 hexamer, highly conserved cytosine in the −17 motif (compared to −13 for other promoters), and moderate A+T content between positions −33 and −18 in the spacer region. Additionally, the discriminator region of these promotors features high A+T content in the first five bases. We identified PficI-17, PficII-33, and PficIII-14 promoters as the optional promoters in the −35 hexamer, spacer region, and discriminator mutation libraries, respectively. These promotors were used as representatives to measure the specific fluorescence and OD600 nm dynamics in different media and to confirm their effect on the expression of different proteins, including egfp (enhanced green fluorescence protein) and rfp (red fluorescence protein). Overall, our findings provide valuable guidance for modifying promoters and developing a promoter library suitable for regulating target genes.

GSH-activated Porphyrin Sonosensitizer Prodrug for Fluorescence Imaging-guided Cancer Sonodynamic Therapy.

Sonodynamic therapy (SDT), which uses ultrasound to trigger a sonosensitizer to generate reactive oxygen species (ROS), is a promising form of cancer therapy with outstanding tissue penetration depth. However, the sonosensitizer may inevitably spread to surrounding healthy tissue beyond the tumor, resulting in undesired side effects under an ultrasound stimulus. Herein, as glutathione (GSH) is overexpressed in the tumor microenvironment, a GSH-activatable sonosensitizer prodrug was designed by attaching a quencher to tetraphydroxy porphyrin for tumor therapy. The prodrug exhibited poor fluorescence and low ROS generation capacity under ultrasound irradiation but it can be activated by GSH to simultaneously switch on fluorescence emission and ROS generation in tumor site. Compared with the non-quenched sonosensitizer, the designed prodrug exhibited significantly higher tumor/healthy organ fluorescence ratios, due to the specific fluorescence and ROS activation by overexpressed GSH in the tumor. Finally, the prodrug exhibited efficient tumor growth inhibition under ultrasound irradiation, further demonstrating its promise as a GSH-activated sonosensitizer prodrug for highly effective cancer treatment.

Debut of a novel AIE-based fluorescent probe as tyrosinase tracer to image skin pigmentary disorders

Tyrosinase (TYR) functions as the critical enzyme in biosynthesis of melanin, and its abnormality is an important hallmark for skin pigmentary disorders. This renders the visualization of tyrosinase dynamics a strategically vital way to facilitate clinical diagnostic and monitoring. However, the use of small molecule fluorescent probes to assess tyrosinase levels and evaluate related therapeutics and drugs has been limited. This is mainly due to the aggregation-induced quenching (ACQ) effects of existing fluorescent probes. In this study, we introduce the first aggregation-induced emission (AIE) small molecule fluorescent probe, BTFTYR, for simple, highly sensitive, and effective detection of tyrosinase activity. The specific fluorescence signal generated by tyrosinase catalytic reaction enables the imaging of pigment cells. We demonstrate its application in visualizing tyrosinase with high contrast in living vertebrates, benefiting from the inherent photobleaching resistance of AIE materials, large Stokes shift, and considerable tissue permeability. We also show the outstanding skin penetration capability of BTFTYR in a skin model. Moreover, we present the first comprehensive evaluation of relevant clinical therapeutic approaches or drugs, including ultraviolet B (UVB) irradiation, kojic acid, and α-MSH, by interpreting the tyrosinase variation with BTFTYR. Finally, our studies confirm that BTFTYR neither causes skin inflammation nor produces visceral toxicity, making it a promising chemical tool for medical diagnosis and drug screening. This tool may further help elucidate the pathomechanisms associated with tyrosinase.

Construction of a novel coordination polymer for selective fluorescence recognition and efficient adsorption of tryptophan in aqueous solution

The efficient separation and purification of L-tryptophan (L-Trp) from fermentation broth are essential for reducing production costs. Here, we report a new 1,1́-binaphthyl-based copper coordination polymer (R-Cu CP) that enables selective fluorescence recognition and efficient adsorption of L-Trp from aqueous solutions. A three-dimensional (3D) CP with a suitable cavity size (17 × 8 Å) was constructed by coordinating copper ions with the dicarboxylate ligand (R)-H3L derived from 1,1́-binaphthyl phosphoric acid. The prepared R-Cu CP exhibits extraordinary adsorption of L-Trp, up to 1055 mg/g. The specific fluorescence quenching response to L-Trp can be achieved by the ultrasonic exfoliation of two-dimensional (2D) CP nanosheets (CPNs). X-ray photoelectron spectroscopy and density functional theory calculations revealed that the efficient adsorption of L-Trp by R-Cu CP is due to the synergistic effect of coordination interactions, hydrogen bonding interactions, electrostatic interactions, and host–guest interactions, and the quenching mechanism is attributed to the photoinduced electron transfer from L-Trp to R-Cu CP and the competitive absorption of excitation energy by L-Trp.

The switchable fluorescent detection of mercury, copper, and iodine in real water samples using bola-amphiphilic-tetraphenylethene

The main objectives of this study are the synthesis of fluorescent bola-amphiphilic-TPE and investigations of its wide range of photochemical properties. Initially, the TSC-TPE-TSC was synthesized for this purpose. Then, using UV–Vis and fluorescence spectroscopy, the AIE studies and interactions of TSC-TPE-TSC with a variety of metal ions were investigated in tap water, distilled water, and EtOH/H2O. The studies revealed that the TSC-TPE-TSC had turn-off sensor characteristics against Hg2+ and Cu2+ ions. Additionally, the interactions of TSC-TPE-TSC, TSC-TPE-TSC-Hg2+, and TSC-TPE-TSC-Cu2+ with anions were also studied in EtOH/H2O (v/v:1, pH 7).The TSC-TPE-TSC-Hg2+ displayed specific turn-on fluorescence characteristics against I¯ ions. Moreover, the computed LOD values for TSC-TPE-TSC/metals (Cu2+ or Hg2+) and TSC-TPE-TSC-Hg2+/I¯ were 2.42 (for Cu2+), 0.39 (for Hg2+), and 3.70 (for I¯), respectively. On the other hand, 1H NMR, FTIR, and DFT experiments were used to determine the binding's morphology. In all reversal studies, the alternate combination of HgCl2 and [Bu4N]I to TSC-TPE-TSC produced switchable off/on variation in all reversal assays. In the end, efficient methods were created in tap water, distilled water, and EtOH/H2O solvent systems for the detection of Hg2+, Cu2+, and I¯.

Cell-Membrane Coated Nanoparticles for Tumor Delineation and Qualitative Estimation of Cancer Biomarkers at Single Wavelength Excitation in Murine and Phantom Models.

Real-time guidance through fluorescence imaging improves the surgical outcomes of tumor resections, reducing the chances of leaving positive margins behind. As tumors are heterogeneous, it is imperative to interrogate multiple overexpressed cancer biomarkers with high sensitivity and specificity to improve surgical outcomes. However, for accurate tumor delineation and ratiometric detection of tumor biomarkers, current methods require multiple excitation wavelengths to image multiple biomarkers, which is impractical in a clinical setting. Here, we have developed a biomimetic platform comprising near-infrared fluorescent semiconducting polymer nanoparticles (SPNs) with red blood cell membrane (RBC) coating, capable of targeting two representative cell-surface biomarkers (folate, αυβ3 integrins) using a single excitation wavelength for tumor delineation during surgical interventions. We evaluate our single excitation ratiometric nanoparticles in in vitro tumor cells, ex vivo tumor-mimicking phantoms, and in vivo mouse xenograft tumor models. Favorable biological properties (improved biocompatibility, prolonged blood circulation, reduced liver uptake) are complemented by superior spectral features: (i) specific fluorescence enhancement in tumor regions with high tumor-to-normal tissue (T/NT) ratios in ex vivo samples and (ii) estimation of cell-surface tumor biomarkers with single wavelength excitation providing insights about cancer progression (metastases). Our single excitation, dual output approach has the potential to differentiate between the tumor and healthy regions and simultaneously provide a qualitative indicator of cancer progression, thereby guiding surgeons in the operating room with the resection process.

A Robust Homogeneous Fluorescence Polarization Immunoassay for Rapid Determination of Erythromycin in Milk.

Erythromycin (ERY) is one of the most common macrolides applied in veterinary medicine to treat diseases or as a feed additive for animal growth promotion. Long-term irrational use of ERY could lead to residues in animal-derived food and the emergence of drug-resistant strains, posing potential threats to human health. In this study, a highly sensitive, specific, robust, and rapid fluorescence polarization immunoassay (FPIA) for the determination of ERY in milk has been described. Herein, to achieve high sensitivity, five tracers of ERY with different fluorescein structures were synthesized and paired with three monoclonal antibodies (mAbs). Under the optimized conditions, the combination of mAb 5B2 and tracer ERM-FITC achieved the lowest IC50 value in the FPIA with 7.39 μg/L for ERM. The established FPIA was used to detect ERY in milk, revealing a limit of detection (LOD) of 14.08 μg/L with recoveries of 96.08-107.77% and coefficients of variations (CVs) of 3.41-10.97%. The total detection time of the developed FPIA was less than 5 min from the addition of samples to the result readout. All the above results showed that the proposed FPIA in this study was a rapid, accurate, and simple method for the screening of ERY in milk samples.

The Role of Tyr-His-Trp Triad and Water Molecule Near the N1-Atom of 2-Hydroperoxycoelenterazine in Bioluminescence of Hydromedusan Photoproteins: Structural and Mutagenesis Study.

Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine, which is tightly bound in the protein inner cavity. The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction. Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed, many features of their functioning are yet unclear. In particular, which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role of the water molecule residing in a proximity to the N1 atom of 2-hydroperoxycoelenterazine in the bioluminescence reaction are still under discussion. With the aim to elucidate the function of this water molecule as well as to pinpoint the amino acid residues presumably involved in the protonation of the primarily formed dioxetanone anion, we constructed a set of single and double obelin and aequorin mutants with substitutions of His, Trp, Tyr, and Ser to residues with different properties of side chains and investigated their bioluminescence properties (specific activity, bioluminescence spectra, stopped-flow kinetics, and fluorescence spectra of Ca2+-discharged photoproteins). Moreover, we determined the spatial structure of the obelin mutant with a substitution of His64, the key residue of the presumable proton transfer, to Phe. On the ground of the bioluminescence properties of the obelin and aequorin mutants as well as the spatial structures of the obelin mutants with the replacements of His64 and Tyr138, the conclusion was made that, in fact, His residue of the Tyr-His-Trp triad and the water molecule perform the "catalytic function" by transferring the proton from solvent to the dioxetanone anion to generate its neutral ionic state in complex with water, as only the decomposition of this form of dioxetanone can provide the highest light output in the light-emitting reaction of the hydromedusan photoproteins.

Variability of fluorescence intensity distribution measured by flow cytometry is influenced by cell size and cell cycle progression

The distribution of fluorescence signals measured with flow cytometry can be influenced by several factors, including qualitative and quantitative properties of the used fluorochromes, optical properties of the detection system, as well as the variability within the analyzed cell population itself. Most of the single cell samples prepared from in vitrocultures or clinical specimens contain a variable cell cycle component. Cell cycle, together with changes in the cell size, are two of the factors that alter the functional properties of analyzed cells and thus affect the interpretation of obtained results. Here, we describe the association between cell cycle status and cell size, and the variability in the distribution of fluorescence intensity as determined with flow cytometry, at population scale. We show that variability in the distribution of background and specific fluorescence signals is related to the cell cycle state of the selected population, with the 10% low fluorescence signal fraction enriched mainly in cells in their G0/G1 cell cycle phase, and the 10% high fraction containing cells mostly in the G2/M phase. Therefore we advise using caution and additional experimental validation when comparing populations defined by fractions at both ends of fluorescence signal distribution to avoid biases caused by the effect of cell cycle and cell size.

Broad-Specificity Screening of Pyrethroids Enabled by the Catalytic Function of Human Serum Albumin on Coumarin Hydrolysis.

Sensing systems based on cholinesterase and carboxylesterase coupled with different transduction technologies have emerged for pesticide screening owing to their simple operation, fast response, and suitability for on-site analysis. However, the broad spectrum and specificity screening of pyrethroids over organophosphates and carbamates remains an unmet challenge for current enzymatic sensors. Human serum albumin (HSA), a multifunctional protein, can promote various chemical transformations and show a high affinity for pyrethroids, which offer a route for specific and broad-spectrum pyrethroid screening. Herein, for the first time, we evaluated the catalytic hydrolysis function of human serum albumin (HSA) on the coumarin lactone bond and revealed that HSA can act as an enzyme to catalyze the hydrolysis of the coumarin lactone bond. Molecular docking and chemical modifications indicate that lysine 199 and tyrosine 411 serve as the catalytic general base and contribute to most of the catalytic activity. Utilizing this enzymatic activity, a broad specific ratiometric fluorescence pyrethroids sensing system was developed. The binding energetics and binding constants of pesticides and HSA show that pyrethroids bind to HSA more easily than organophosphates and carbamates, which is responsible for the specificity of the sensing system. This study provides a general sensor platform and strategy for screening pesticides and reveals the catalytic activity of HSA on the hydrolysis of the coumarin lactone bond, which may open innovative horizons for the chemical sensing and biomedical applications of HSA.

Simulation monitoring of tetracyclines in wastewater based on fluorescence image processing and machine learning classifier

Antibiotics, as an emerging pollutant, have attracted extensive attention due to their trace residues and toxic side effects in the environment. Here, we constructed a new detection mechanism for tetracyclines in sewage by online trace sensing through machine learning (ML) algorithm and image fingerprint extraction method. The image fingerprint information was obtained from the blue to red specific fluorescence response generated by the interaction between the luminescent metal-organic framework (MOF) with tetracyclines, which greatly improved the ability against background interference. Fluorescence images of samples with different concentrations were collected, and the RGB mean value of sample images was extracted through the program as "fingerprint information". Then k-means clustering model (KCM) and hierarchical clustering model (HCM) were constructed to analyze the differences in fingerprint information of samples with different concentrations by clustering analysis method in machine learning. The clustering results showed good clustering characteristics of samples with similar concentrations, which provided reliable support for rapid detection while avoiding the use of expensive large instruments and time-consuming analysis methods.

A “turn-on” fluorescent sensor for Pb2+ detection based on nitrogen doping carbon dots

Nitrogen-doped carbon dots (N-CDs) were synthesized via a green chemistry route and subsequently employed as a fluorescent sensing probe for Pb2+ ions. Through a series of characterizations, the properties of the obtained N-CDs were comprehensively deciphered and investigated. In particular, the specific fluorescence response for Pb2+ was obviously promoted by a “turn-on” mode, even in the presence of other interfering metal ions. According to the established N-CDs sensors, a linear correlation of fluorescence intensity and Pb2+ concentration was formed in the range of 0∼50 μM. This sensing platform displayed a sensitive and selective detection towards Pb2+ ions, in which a limit of detection (LOD) was achieved up to 0.92 μM. Finally, the underlying sensing mechanism towards Pb2+ was proposed by a photo-induced electron transfer (PET) inhibition process, which was demonstrated by a density functional theory (DFT) simulation study. This work provides a promising platform for the analysis of Pb2+ ions in aqueous environmental samples.

Estimation of soil porosity in mine tailing using parameters from instrumented oedometer test

In the geological field, unexpected tailing failures have recently occurred and caused critical damage. The objective of this study was to evaluate the porosity of mine tailing using compressibility and elastic wave velocities from an instrumented oedometer test. Three samples (MS1, MS2, MS3) were obtained from the mine tailing at different depths, and their properties were assessed. The oedometer test was conducted from 0 to 1280 kPa for MS1, MS2 (with various initial stresses), and MS3, and the elastic wave velocities were measured during each loading and unloading step. The specific gravity, X-ray fluorescence, and X-ray diffraction test results revealed a portion of hematite (Fe2O3) in all the samples. According to the instrumented oedometer test, the compressibility and elastic wave velocities of the samples varied with respect to the sampling depth and stress conditions. In particular, the α factor and β exponent from the shear wave velocity followed the empirical relationship of general soil types. Empirical porosity evaluation methods reliably estimated the sample porosity at shallow depths by using the parameters from the instrumented oedometer tests. Thus, the in situ porosity at a mine tailing can be effectively evaluated if the profiling of elastic velocities in the field is available.

Electronic tuning strategy in H2S-triggered fluorescence sensing and subsequent photodynamic therapy of colon cancer

The molecular probe oriented detection of disease-associated hallmarks has aroused great attention in analytical domain. However, the bioactivity evaluation of probe after recognition is rarely investigated in detection process. Herein, a rational strategy by electronic tuning for specific H2S-activated fluorescence sensing and in situ thio-photosensitizer generation for enhanced solid tumor phototherapy was proposed. Three NIR-absorbing molecular probes (Cy735, Cy795 and Cy841) were synthesized based on our strategy. Different electronic distribution in three probes significantly changed the reactive site of H2S-associated thiolysis. For Cy735, further initiate in situ formation of thio-photosensitizer Cy581, which diminish the energy gap of S1-T1, promote the process of intersystem crossing (ISC), thus resulting in high singlet oxygen (1O2) generation efficiency and PDT therapeutic effect. Cy735 and Cy795 could successfully be employed for the fluorescence imaging of endogenous H2S in colon cancer cells. The therapeutic evaluation exhibited that Cy735 can strikingly promote tumorous cell death and suppress solid tumor growth under 808 nm light irradiation after H2S stimulated. In vivo model revealed that, the fluorescence signal of Cy735 in the tumor site was obviously enhanced in 12–32 h after administered through tail intravenous injection, which indicated that Cy735 can selectively target and accumulate in the solid tumor and the retention time is about 20 h. It is adequately envisioned that our strategy will provide promising tool for efficient tumor photoablation as well as extend the options of excellent agents for clinical cancer therapy.

Di-(2-ethylhexyl) phthalate impairs angiogenesis and hematopoiesis via suppressing VEGF signaling in zebrafish

Di-(2-ethylhexyl) phthalate (DEHP) is among the most widely used plasticizers in plastic production, which has been detected in various environments. However, DEHP safety remains poorly known. Using zebrafish models, the effects of DEHP on the angiogenesis and hematopoiesis, and the underlying mechanism, were studied. Transgenic zebrafish embryos with specific fluorescence of vascular endothelial cells, myeloid cells, or hematopoietic stem cells were exposed to 0, 100, 150, 200, or 250 nM of DEHP for 22, 46 or 70 h, followed by fluorescence observation, endogenous alkaline phosphatase activity measurement, erythrocyte staining, and gene expression analysis by quantitative PCR and whole mount in situ hybridization. High DEHP concentrations decreased the sprouting rate, average diameter, and length, and the expansion area of the vessels lowered the EAP activity and suppressed the vascular endothelial growth factor (vegf) and hematopoietic marker genes, including c-myb, hbae1, hbbe1, and lyz expressions. DEHP treatment also decreased the number of hematopoietic stem cells, erythrocytes, and myeloid cells at 24 and 72 hpf. These DEHP-induced angiogenetic and hematopoietic defects might be alleviated by vegf overexpression. Our results reveal a plausible mechanistic link between DEHP exposure-induced embryonic angiogenetic defect and hematopoietic impairment.