Peptide-based Fluorescent Probe Research Articles

Rational design of peptide-based fluorescent probe for sequential recognitions of Cu(II) ions and glyphosate: Smartphone, test strip, real sample and living cells applications

A new peptide-based fluorescent probe named DMDH with easy-to-synthesize, excellent stability, good water solubility and large Stokes shift (225 nm) was synthesized for highly selective sequential detections of copper ions (Cu2+) and glyphosate (Glyp). DMDH demonstrated great detection performance towards Cu2+via strong fluorescence quenching, and forming non-fluorescence DMDH-Cu2+ ensemble. As a new promising cascade probe, the fluorescence of DMDH-Cu2+ ensemble was significantly recovered based on displacement approach after glyphosate was added. Interestingly, the limit of detections (LODs) for Cu2+ and glyphosate were 40.6 nM and 10.6 nM, respectively, which were far lower than those recommended by the WHO guidelines for drinking water. More importantly, DMDH was utilized to evaluate Cu2+ and glyphosate content in three real water samples, demonstrating that its effectiveness in water quality monitoring. Additionally, it is worth noting that DMDH was also applied to analyze Cu2+ and glyphosate in living cells in view of significant cells permeability and low cytotoxicity. Moreover, DMDH soaked in filter paper was used to create qualitative test strips and visually identify Cu2+ and glyphosate through significant color changes. Furthermore, smartphone RGB color recognition provided a new method for semi-quantitative testing of Cu2+ and glyphosate in the absence of expensive instruments.

Universal organophosphate pesticides detection by peptide based fluorescent probes

In practical applications, the rapid and efficient detection of universal organophosphorus pesticides (OPs) can assist inspectors in quickly identifying the presence of OPs in samples. However, this presents a challenge for most well-established methods, typically designed to detect only a specific type of organophosphorus molecule at a time. In this proof-of-concept study, we draw inspiration from the structural similarities among OPs to develop innovative peptide-based fluorescence probes for the first time, which could efficiently detect a broad range of OPs within a mere 3 min. Analysis of fluorescence curve fitting reveals a clear linear correlation between the fluorescent intensity of the peptide probes and the concentration of OPs. Additionally, the selectivity analysis indicates that these peptide fluorescent probes exhibit an excellent response to various OPs while maintaining sufficient selectivity for detecting other pesticide types. Accurate sample analysis has also highlighted the potential of these peptide probes as practical tools for the rapid detection of OPs in actual vegetable samples. In summary, this proof-of-concept study presents an innovative approach to designing and developing ultrafast, universally peptide-based OP probes. These custom-designed peptide probes may facilitate rapid sample screening and offer initial quantification for OPs, potentially saving valuable time and effort in practical OP detection.

Ratiometric peptide-based fluorescent probe with large Stokes shift for detection of Hg2+ and S2− and its applications in cells imaging and smartphone-assisted recognition

In this work, a new ratiometric fluorescent probe DKA was synthesized based on the double sides of lysine backbone conjugated with alanine and dansyl groups. DKA exhibited fluorescence ratiometric response for Hg2+ with high sensitivity (13.4 nM), specific selectivity (only Hg2+), strong anti-interference ability (no interference), fast recognition (within 60 s) and wide pH range (5–10). The stoichiometry of binding of DKA and Hg2+ was determined to be 1:1 via Job’s plot, ESI-HRMS and 1HNMR titration analysis. Subsequently, the in situ formation of DKA-Hg2+ complex was used for highly selective detection of S2− as a novel fluorescence “on–off” probe, and the lowest detection limit for S2− was 12.9 nM. In addition, DKA possessed excellent cells permeation and low toxicity, and fluorescence imaging of Hg2+ and S2− was performed in living Hacat cells. Most importantly, the digital imaging using a smartphone color recognition APP indicated that DKA could semi-quantitatively and visually detected Hg2+ and S2− without expensive equipment.

Novel Peptide-Based Fluorescent Probe for Simultaneous Sensing of Chymotrypsin and Hydrogen Peroxide.

The developed multifunctional fluorescent probe enables the simultaneous detection of chymotrypsin as a model protease and hydrogen peroxide as a representative of reactive oxygen species (ROS) in biologically relevant concentration ranges. The chymotrypsin sensing is based on the cleavage of its selectively recognizable peptide sequence and the consequent disruption of FRET between coumarin (DEAC) and fluorescein (FL). Analogously, the presence of hydrogen peroxide causes the gradual degradation of the H2O2-labile benzopyrylium-coumarin (BC) dye. Considering the fluorescence emission responses of individual chymotrypsin-peroxide probe-attached fluorophores after their excitation at 425 nm, the sole presence of either chymotrypsin (50-1000 ng/mL) or hydrogen peroxide (10-200 μM) in a sample could be unambiguously confirmed or refuted. In addition, reliable simultaneous detection and approximate quantification of both studied species in the concentration ranges of 100-1000 ng/mL and 20-200 μM for chymotrypsin and H2O2, respectively, could be performed as well. The obtained results are summarized and visualized in the graphical models.

A novel ratiometric peptide-based fluorescent probe for sequential detection of Hg2+ and S2− ions and its application in living cells and zebrafish imaging

A new ratiometric peptide-based fluorescent probe DWPH was designed and synthesized, comprising dansyl fluorophore as a fluorescent dye, and tripeptide backbone (Trp-Pro-His-NH2) as a recognition group. The addition of Hg2+ caused the maximum emission peak of DWPH to blue shift from 560 nm to 510 nm. DWPH exhibited large Stokes shift (230 nm), satisfactory water solubility (100 % aqueous medium), good selectivity (only Hg2+), high sensitivity (24.6 nM), rapid response (within 50 s) and strong anti-interference ability for Hg2+ detection over a wide pH range (7–11). Additionally, the complex DWPH-Hg2+ as a relay response probe could also be applied to S2− according to displacement approach. Notably, the detection limit for S2− was calculated as 23.3 nM, exhibiting that DWPH showed great potential for environmental monitoring and bioimaging. In addition, DWPH were successfully used to determine Hg2+ and S2− in living cells and zebrafish based on excellent permeability and low cytotoxicity. What’s more, the gradient concentration color changes of Hg2+ and S2− were combined with the smartphone APP to obtain red–green–blue (RGB) values, thus enabling rapid semi-quantitative detection of Hg2+ and S2− without expensive instruments.

A novel AIE peptide-based fluorescent probe for highly selective detection of mercury(II) ions and its application in food samples and cell imaging

A novel peptide-based fluorescent probe TPE-PGH was firstly developed based on aggregation-induced emission (AIE) effect. The fluorescence intensity of TPE-PGH was largely ignored in the low water fraction, whereas bright blue fluorescence was observed in the high water fraction. TPE-PGH achieved high selectivity and rapid detection of Hg2+, and has excellent sensitivity to detect Hg2+ with low LOD (58.1 nM). Interestingly, TPE-PGH provided a significant color changed from dark blue to bright cyan upon binding with Hg2+ ions under UV lamp (365 nm). In addition, TPE-PGH was able to simultaneously visualize Hg2+ in living cells with different fluorescence intensities based on excellent cells permeability and low toxicity. Further, TPE-PGH exhibited high precision (RSD < 4 %), and was successfully applied for detecting Hg2+ in real food samples, such as milk, soda drink, red wine, cucumber, tomato and carrot. Successful test strips responses also demonstrated that the potential of TPE-PGH for portable detection of Hg2+ in real samples. Especially, AIE based-test strips soaked in different Hg2+ concentrations was successfully utilized for semi-quantitative detection of Hg2+ using the RGB APP installed on a smartphone, which will provide a new approach for smart and portable detection of contaminated heavy metal ions in food samples.

A novel peptide-based fluorescent probe with large Stokes shift for simultaneous detection of zinc (II) and mercury (II): Smartphone, test strips, living cells and real samples applications

A simple and efficient peptide-based fluorescent probe L with large Stokes shift was synthesized, which used dansyl group as fluorophore and tetrapeptide backbone (HisGluPheCys-NH2) as recognition group for the determinations of Zn2+and Hg2+by different fluorescence response modes in 100% aqueous solution. L exhibited a highly efficient and specific selectivity for Zn2+ based on fluorescence enhancement as well as the naked eye recognition of Zn2+ was realized through the solutions color changed. Meanwhile, as a highly selective “turn off” probe, L could rapidly detect Hg2+ based on obvious fluorescence quenching. The limit of detection (LODs) for Zn2+ and Hg2+were 52.6 and 49.7 nM, respectively. Moreover, L has good accuracy, exciting water-solubility and low toxicity, which was successfully applied to determinations of Zn2+ and Hg2+ in real water samples and fluorescence imaging of Zn2+ and Hg2+ in living cells. Test strips of probe L was prepared by immersion and used for visual detection of Zn2+ and Hg2+by naked eye. Finally, the gradient change of fluorescence color of L was captured by smartphone, and then content analysis was carried out by smartphone color recognition APP.

Development of ratiometric fluorescent probes based on peptides for sensing Pb2+ in aquatic environments and human serum.

The detection of Pb2+ ions in aquatic environments and biofluid samples is crucial for assessment of human health. Herein, we synthesized two fluorescent probes (1 and 2) consisting of the peptide receptor for Pb2+ and a benzothiazolyl-cyanovinylene fluorophore that exhibited excimer-like emission when it aggregated. The peptide-based probes sensitively detected Pb2+ in purely aqueous solution (1% DMF) through ratiometric fluorescent response with a decrease in monomer emission at 520nm and an increase in excimer emission at 570nm. Specially, probe 2 showed remarkable detection features such as high selectivity for Pb2+over 15 metal ions, high binding affinity (Kd=5.83×10-7 M) for Pb2+, significant emission intensity changes, low detection limit (3.8nM) of Pb2+, high water solubility, and visible light excitation (450nm). Probe 2 was successfully used to quantify nanomolar concentration (0∼800nM) of Pb2+ in real water samples (ground water and tap water). Specially, 2 was successfully applied for the quantification of Pb2+ in human serum by combination of microwave-assisted human serum digestion and filtration of digested serum by anion exchange cartridge. We clearly investigated the binding mode of 2 with Pb2+ using 1H NMR, IR spectroscopy, pH titration, confocal microscopy, and size analysis. The peptide-based fluorescent probe might have great application potential for sensing Pb2+ in aquatic environments and biofluid samples.

Self-Assembly and Disassembly of Membrane Curvature-Sensing Peptide-Based Deep-Red Fluorescent Probe for Highly Sensitive Sensing of Exosomes.

With increasing knowledge of the diverse roles of exosomes in biological processes, much attention has been paid to the development of analytical methods for exosome analysis. Here, we developed a new class of amphipathic helical (AH) peptide-based fluorescent probes for highly sensitive detection of exosomes in a mix and read manner. Membrane curvature-sensing AH peptide (ApoC) was coupled with lipophilic tail (C12)-carrying thiazole red (TR) for construction of a self-assembly/disassembly based fluorescence "off-on" sensing system for target exosomes. ApoC-TRC12 has extremely weak emission due to the formation of the aggregates, whereas it becomes emissive in response to the target exosomes through the binding-induced disassembly of ApoC-TRC12. We demonstrated that the C12 unit attached to the TR unit had a favorable effect on both fluorescence response (signal-to-background: S/B) and binding affinity. ApoC-TRC12 was applicable to rapid and simple detection of exosomes with high detection sensitivity (limit of detection ≈ 103 particles/μL).

A novel fluorescent probe based on a tripeptide-Cu(II) complex system for detection of histidine and its application on test strips and smartphone

Herein, we reported a novel peptide-based fluorescent probe DSSH for highly selective and sensitive detections of both Cu2+ and l-histidine (l-His). DSSH exhibited different color changes and fluorescence “on–off” response toward Cu2+ with a 2:1 binding stoichiometry, and the limit of detection (LOD) for Cu2+ was calculated to be 22.9 nM. The in situ formed DSSH-Cu2+ ensemble showed obvious fluorescence “off–on” response to l-His based on replacement reaction with Cu2+, as well as the discernable color changes under 365 nm UV lamp irradiation with “naked eye”. The specificity of Cu2+/l-His interactions allowed l-His to be determined without interference from other amino acids, and the detection limit of DSSH-Cu2+ ensemble response to l-His was determined as 25.7 nM. Notably, DSSH was successfully applied for detecting Cu2+ and l-His in RKO living cells owing to its remarkable fluorescence behavior and low cytotoxicity. Test strips experiments suggested that DSSH can recognize Cu2+ and l-His together by a remarkable fluorescence change. More importantly, smartphone was combined with l-His solutions of different concentrations and converted into digital values through RGB channels, which was successfully used for semi-quantitative identification of l-His, and the limit of detection (LOD) was 0.97 μM.

Glucose-triggered self-assembly of peptide-based fluorescent probes for highly selective ratiometric detection of D-Glucose in aqueous solution at physiological pH and biological fluids

D-Glucose (Glu) is the principal source of energy production for important organs in human beings. We have first developed a monoboronic acid-based fluorescent sensor (1) for highly selective ratiometric detection of Glu among hexose in aqueous solution at physiological pH. The peptide-based probe bearing a pyrene fluorophore and a phenylboronic acid showed remarkable sensing ability for Glu such as high sensitivity for Glu, high enhancement (70-fold) of the excimer emissions, low detection limit (3.1 µM), fast response (< 10 mins), and high-water solubility. The fluorescent study of peptide-based probes (1−5) suggested that the amphiphilic peptide moiety consisting of the positively charged amino acid (Arg and Lys) and hydrophobic Trp residue played a critical role for the ratiometric response toward Glu. The binding mode study of 1 indicated that the probe formed a 2:1 complex with Glu and then the complexes self-assembled, resulting in significant enhancement of excimer emission, whereas the probe formed a 1:1 complex with Fru that did not assemble, resulting in no considerable fluorescent changes. We demonstrated that Glu induced the formation of nanoparticles and supramolecular gels of 1 depending on the concentration of the probe and Glu. 1 could quantify Glu in real urine and human serum samples by ratiometric response. This was the first example of self-assembling peptide-based probe triggered by Glu for fluorescent detection of Glu and hydrogel formation. Glu-triggered self-assembly of the peptide-based probes could be applied not only for novel sensing systems but also for the development of drug delivery and regenerative medicine.

A novel peptide-based relay fluorescent probe with a large Stokes shift for detection of Hg2+ and S2− in 100 % aqueous medium and living cells: Visual detection via test strips and smartphone

Herein, a novel relay peptide-based fluorescent probe DGRK was synthesized via solid phase peptide synthesis (SPPS) technology. DGRK exhibited excellent water-solubility, good stability, remarkably large Stokes shift (230 nm) and high selectivity response to Hg2+ with a non-fluorescence complex DGRK-Hg2+ formation via a 1:1 binding mode. Further studies indicated that the DGRK-Hg2+ complex could act as a secondary probe for rapidly and sequentially detecting S2− based on fluorescent “off–on” response, and without interference from a range of anions. The limit of detection (LOD) for Hg2+ and S2− were calculated to be 33.6 nM and 60.9 nM, respectively. In addition, The reversibility of interaction of confirmed that the continuous and reversible recognition behavior of Hg2+ and S2− by the probe DGRK, and could be cycled more than 5 times. In addition, DGRK could be successfully applied to the fluorescence imaging of Hg2+ and S2− in two living cells based on excellent cells permeability and low cytotoxicity. Meanwhile, DGRK was successfully used to create the low-cost and portable test strips for visual detection and rapid analysis under 365 nm UV lamp, and the test strips combined with a smartphone (RGB color) was successfully applied to the semi-quantitative analysis and monitoring of dynamic changes of Hg2+ levels.

Peptide-based fluorescent and colorimetric dual-functional probe for visual detection of Cu2+, Hg2+ and S2− in 100% aqueous media, living cells and paper test strips

In this study, a novel peptide-based fluorescent and colorimetric dual-functional probe (FAHK) was designed and synthesized by simulating the structure of copper peptide. FAHK exhibited high selectivity and rapid response to Cu2+ and Hg2+ with two non-fluorescence ensembles FAHK-Cu2+ and FAHK-Hg2+ formation by the 2:1 coordination mode, and the limits of detection for Cu2+ and Hg2+ were 37.1 nM and 20.8 nM, respectively. FAHK showed a naked eye colorimetric response for Cu2+ and Hg2+ in an aqueous solutions under visible light. As two new promising cascade probes, FAHK-Cu2+ and FAHK-Hg2+ ensemble were used for sequential selective recognition of S2− according to the displacement approach with fluorescence “turn-on” response and colorimetric change, and the limit of detection were 0.27 μM (FAHK-Cu2+ ensemble) and 0.12 μM (FAHK-Hg2+ ensemble) for S2−, which were well below the safety level the H2S standard in drinking waters (14.8 µM, WHO guidelines). What’ more, in view of low toxicity and biocompatibility, the potential application of FAHK in biological systems was demonstrated via living cells imaging. More importantly, FAHK could also be made into the paper test strips for visual detection of Cu2+, Hg2+ and S2− with the naked eye under 365 nm UV light and visible light, which suggested that better portability and operation simplicity that is favourable for on-site analysis.

Based on lapatinib innovative near-infrared fluorescent probes targeting HER1/HER2 for in vivo tumors imaging

Human epidermal growth factor receptors HER1 and HER2, overexpressed in non-small cell lung cancer, colorectal cancer, liver cancer, are key regulators of tumor cells proliferation, invasion and survival. Many antibody- or peptide-based fluorescent probes targeted to HER1/HER2 are under active clinical evaluation. However, the effective small-molecule near-infrared (NIR) fluorescent probes are still lacking. Herein, with the strategy of drug repurposing, we developed a series of HER1/HER2-targeted probes YQ-H (01–07) composed of fluorophore (a cyanine dye, MPA), linker unit and targeted unit (lapatinib, LAP). The synthesized probes were evaluated in vitro and in vivo tumor specificity/affinity. Specially, the probe YQ-H-06 exhibited optimal pharmacokinetic property and tumor/normal tissue ratio (T/N) in tumor-bearing mice. Furthermore, we evaluated the targeting capability of YQ-H-06 in orthotopic colorectal cancer and orthotopic hepatic carcinoma mice. It was indicated that YQ-H-06 had the characteristic of great biosafety, favorable pH and chemical stability, as well as provided excellent tumor contrast in orthotopic murine tumor models. The NIR fluorescent probe YQ-H-06 will shed light on tumor detection and fluorescence-guided surgery.

Cover Picture: A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity (Angew. Chem. Int. Ed. 30/2022)

Sylite, a short, dimerized peptide-based fluorescent probe, binds gephyrin, a hallmark protein of the inhibitory synapse. As reported by Christian G. Specht, Hans M. Maric et al. in their Research Article (e202202078), Sylite visualizes inhibitory synapses both in neuron cultures and brain tissue and can be flexibly applied for widefield and confocal 3D volumetric synapse visualization, for brain tissue inhibitory circuits mapping and for super-resolution imaging of synapses. The cover picture shows a stylized image of a mouse hippocampus in coronal section stained with Sylite in green and DAPI nuclear stain in blue. Cover design by Christian Günther.

A novel peptide-based fluorescent probe for highly selective detection of mercury (II) ions in real water samples and living cells based on aggregation-induced emission effect

A new fluorescent probe TPE-GHK was synthesized containing a tetrastyrene (TPE) derivative as fluorophore and classical tripeptide (Gly-His-Lys-NH2) as a receptor based on the aggregation-induced emission (AIE) mechanism. TPE-GHK displayed high selectivity and rapid fluorescent "turn-on" response to Hg2+ among other competitive metal ions. The 2:1 complex binding mechanism of TPE-GHK toward Hg2+ was verified by fluorometric titration, Job's plots, and ESI-HRMS spectra. The fluorescent emission showed a good linear response in the range of 0-1.0 μM with the low detection limit of 28.6 nM. Meanwhile, TPE-GHK exhibited the excellent biocompatibility and low toxicity and was successfully applied in monitoring Hg2+ in living CAKI 2 cells, which demonstrated its potential application in environment and biological science. More importantly, TPE-GHK could be used to detect Hg2+ in two real water samples and also was successfully designed as test strips.

A Method ofTumor In Vivo Imaging with a New Peptide-Based Fluorescent Probe.

Precise surgical resection directly influences the prognosis and survival of patients with solid tumors. However, it is often difficult to distinguish tumor from normal tissue during resection without any intraoperative imaging guidance. Image-guided surgery particularly when coupled with a near-infrared (NIR) fluorescent agent may improve positive-margin rate thereby improving the overall prognosis. We have developed a unique tumor-targeting fluorescence imaging agent that can aid in the accurate localization of human cancer cells in preclinical settings. The NIR imaging agent, ICG-p28, a water-soluble, nontoxic, and pan-tumor targeting probe consisting of a cell-penetrating peptide (p28) conjugated to indocyanine green (ICG), can accurately localize tumors in vivo. Development of the noninvasive, targeted imaging agent can potentially improve in the resections of tumors by enabling the localization of lesions that are currently difficult or impossible to detect by visual observation or palpation. Here, we describe the methodsof preclinical animal imaging models by using NIR fluorescence imager coupled with a new tumor-targeting agent.

Peptide-Based Fluorescent and Colorimetric Dual-Functional Probe for Visual Detection of Cu2+, Hg2+ and S2− in 100% Aqueous Media, Living Cells and Paper Test Strips

Peptide-Based Fluorescent and Colorimetric Dual-Functional Probe for Visual Detection of Cu2+, Hg2+ and S2− in 100% Aqueous Media, Living Cells and Paper Test Strips

A bifunctional peptide-based fluorescent probe for ratiometric and “turn-on” detection of Zn(II) ions and its application in living cells

In this work, a bifunctional peptide-based fluorescent probe L containing a tetrapeptide scaffold (Pro-Gly-His-Trp-NH2) and a dansyl group was synthesized using solid phase peptide synthesis (SPPS) technology. As designed, L, based on a FRET mechanism, exhibited high selectivity, excellent ratiometric signals, and fast response to Zn2+ in aqueous solutions at an excitation wavelength of 280 nm. In addition, when excited at 320 nm, L exhibited a fluorescent “turn-on” response towards Zn2+ based on PET mechanism. More importantly, the stoichiometry of L and Zn2+ was determined to be 2:1 by fluorescent titration, Job’s plot method, and ESI-MS spectrometry. The association constant for Zn2+ ions was determined to be 6.26 × 108 M−2, while the limit of detection (LOD) of L was estimated as 5.43 nM, which is a much lower value than WHO and EPA guidelines for drinking water. Moreover, L was successfully applied to detect both Zn2+ and Cu2+ in living cells due to good biocompatibility and excellent low toxicity.

A novel peptide-based fluorescent probe with a large stokes shift for rapid and sequential detection of Cu2+ and CN− in aqueous systems and live cells

A novel fluorescent probe (DSD) was reasonably designed and synthesized with dansyl-labeled dipeptide (Dan-Ser-Asp-NH2). DSD featured remarkably large Stokes shift (230 nm) and perfect water solubility, and exhibited high selectivity and rapid recognition toward Cu2+via fluorescence quenching. The detection limit of DSD for Cu2+ was 2.4 nM, indicated that DSD has excellent sensitivity. In addition, the stoichiometry between DSD and Cu2+ were detected as 1:1 by fluorescence titration, Job’s plot and ESI-HRMS data. As designed, DSD-Cu2+ system was able to sequentially detect CN− according to the displacement approach with fluorescence “off–on” response, and the detection limit for CN− was calculated to be 41.9 nM. Specifically, the response time of DSD with Cu2+ and CN− was less than 40 s, which rendered it suitable for real time detection in actual water samples. In addition, with the alternate addition of Cu2+ and CN−, the reversible cycles could be repeated for at least 10 times, indicated that DSD was a promising reversibility probe. DSD showed low toxicity and good biocompatibility, and was successfully applied to detect Cu2+ and CN− in living cells.