•Comprehensive review on site-accurate bioconjugation reactions.•Comprehensive review on proximity-induced covalent reactions.To achieve precise control of the signaling events or to achieve unmistakable synthesis of biomolecules, nature has evolved organic reactions involving proteinogenic amino acids with unparalleled site selectivity. For example, dedicated enzymes accurately dictate the site of post-translational modifications in signaling proteins, and ribosomes precisely link the C-terminal carboxylic acid of one unprotected amino acid with the N-terminal amino group of the other amino acid through spatially confined proximity. For many years, chemists have been striving to achieve site selectivity on biomolecules by mimicking nature. Driven by the development of chemoselective protein conjugation reactions, enzymology and protein–protein interactions, the past decade has witnessed a boom in site-selective protein conjugation reactions. (In this review, a site-selective protein conjugation reaction is defined as an organic reaction that targets a single amino acid instead of a kind of amino acids in a protein or a proteome under physiological conditions, for example, a single cysteine residue among all of the cysteines.) In this review, we summarize the recent advancements of bioconjugation reactions that demonstrate this feature of precise site selectivity, focusing on the reactions of the proteinogenic amino acids (excluding those at non-coded or non-proteinogenic amino acids that are introduced to proteins through genetic manipulations).Download high-res image (171KB)Download full-size image

Alkaline phosphatase (ALP) is a family of enzymes involved in the regulation of important biological processes such as cell differentiation and bone mineralization. Monitoring the activity of ALP in serum can help diagnose a variety of diseases including bone and liver diseases. There has been growing interest in developing new chemical tools for monitoring ALP activity in living systems. Such tools will help further delineate the roles of ALP in biological and pathological processes. Previously reported fluorescent probes has a number of disadvantages that limit their application, such as poor selectivity and short-wavelength excitation. In this work, we report a new two-photon fluorescent probe (TP-Phos) to selectively detect ALP activity. The probe is composed of a two-photon fluorophore, a phosphate recognition moiety, and a self-cleavable adaptor. It offers a number of advantages over previously reported probes, such as fast reaction kinetics, high sensitivity and low cytotoxicity. Experimental results also showed that TP-Phos displayed improved selectivity over DIFMUP, a commonly utilized ALP probe. The selectivity is attributed to the utilization of an ortho-functionalised phenyl phosphate group, which increases the steric hindrance of the probe and the active site of phosphatases. Moreover, the two-photon nature of the probe confers enhanced imaging properties such as increased penetration depth and lower tissue autofluorescence. TP-Phos was successfully used to image the endogenous ALP activity of hippocampus, kidney and liver tissues from rat.

The development of novel antimicrobial materials with high antimicrobial activity and low environmental impact is of importance for global health, but has proven to be challenging. Herein we report a facile mineralization process to create a flower-like, porous antimicrobial agent, which is stable, selective, effective and environmentally benign. This new antimicrobial material is made of organic polydopamine (PD) and inorganic (copper phosphate) components, where the incorporation of PD on the hybrid architecture endows the direct in situ reduction of silver ions into silver nanoparticles (Ag NPs) without the need of external toxic reductants. The combination of Ag NPs and high surface area of the nanoflower results in high selectivity in the antimicrobial activity towards Gram-negative Escherichia coli (E. coli), while leaving co-cultured mammalian cells healthy and intact. Moreover, we show that the hybrid antimicrobial material is stable, and can be easily recovered after use, avoiding the persistent hazard to the environment. We envision that this novel antimicrobial agent may find useful applications for clinical studies and industrial products.

A facile fluorescent probe (NBD-DOP) has been developed to detect hypochlorous acid (HOCl) in this study. The probe consists of a NBD fluorophore and a dopamine moiety that reacts with HOCl specifically. The dopamine group quenches the fluorescence of NBD efficiently through a photoinduced electron transfer (PET) effect. Experimental data showed that NBD-DOP could detect HOCl with ultrafast response, high sensitivity and high selectivity in a wide pH range. The probe could also be used to detect the myeloperoxidase enzyme that produces HOCl. Moreover, NBD-DOP has been applied in the imaging of exogenous and endogenous HOCl in living cells by confocal fluorescence microscopy.

Alkaline phosphatases are a group of enzymes that play important roles in regulating diverse cellular functions and disease pathogenesis. Hence, developing fluorescent probes for in vivo detection of alkaline phosphatase activity is highly desirable for studying the dynamic phosphorylation in living organisms. Here, we developed the very first reaction-based near-infrared (NIR) probe (DHXP) for sensitive detection of alkaline phosphatase activity both in vitro and in vivo. Our studies demonstrated that the probe displayed an up to 66-fold fluorescence increment upon incubation with alkaline phosphatases, and the detection limit of our probe was determined to be 0.07 U/L, which is lower than that of most of alkaline phosphatase probes reported in literature. Furthermore, we demonstrated that the probe can be applied to detecting alkaline phosphatase activity in cells and mice. In addition, our probe possesses excellent biocompatibility and rapid cell-internalization ability. In light of these prominent properties, we envision that DHXP will add useful tools for investigating alkaline phosphatase activity in biomedical research.Keywords: alkaline phosphatase; bioimaging; fluorescent probe; mice; near-infrared;

•A reaction-based sensor for Cu2+ detection with NIR excitation and emission.•The detection limit for Cu2+ is as low as 29 nM in aqueous buffer.•The probe shows special selectivity for Cu2+ over other metal ions.Copper (II) is one of the most of important cofactors for numerous enzymes and has captured broad attention due to its role as a neurotransmitters for physiological and pathological functions. In this article, we present a reaction-based fluorescent sensor for Cu2+ detection (NIR-Cu) with near-infrared excitation and emission, including probe design, structure characterization, optical property test and biological imaging application. NIR-Cu is equipped with a functional group, 2-picolinic ester, which hydrolyzes in the presence of Cu2+ with high selectivity over completed cations. With the experimental conditions optimized, NIR-Cu (5 μM) exhibits linear response for Cu2+ range from 0.1 to 5 μM, with a detection limit of 29 nM. NIR-Cu also shows excellent water solubility and are highly responsive, both desirable properties for Cu2+ detection in water samples. In addition, due to its near-infrared excitation and emission properties, NIR-Cu demonstrates outstanding fluorescent imaging in living cells and tissues.

Histone deacetylases (HDACs) play important roles in regulating various physiological and pathological processes. Developing fluorescent probes capable of detecting HDAC activity can help further elucidate the roles of HDACs in biology. In this study, we first developed a set of activity-based fluorescent probes by incorporating the Kac residue and the O-NBD group. Upon enzymatic removal of the acetyl group in the Kac residue, the released free amine reacted intramolecularly with the O-NBD moiety, resulting in turn-on fluorescence. These designed probes are capable of detecting HDAC activity in a continuous fashion, thereby eliminating the extra step of fluorescence development. Remarkably, the amount of turn-on fluorescence can be as high as 50-fold, which is superior to the existing one-step HDAC fluorescent probes. Inhibition experiments further proved that the probes can serve as useful tools for screening HDAC inhibitors. Building on these results, we moved on and designed a dual-purpose fluorescent probe by introducing a diazirine photo-cross-linker into the probe. The resulting probe was not only capable of reporting enzymatic activity but also able to directly identify and capture the protein targets from the complex cellular environment. By combining a fluorometric method and in-gel fluorescence scanning technique, we found that epigenetic readers and erasers can be readily identified and differentiated using a single probe. This is not achievable with traditional photoaffinity probes. In light of the prominent properties and the diverse functions of this newly developed probe, we envision that it can provide a robust tool for functional analysis of HDACs and facilitate future drug discovery in epigenetics.

A simple molecule, tetrafluoroterephthalonitrile (4F-2CN), was discovered to be an efficient fluorescent probe for detecting biological thiol species. The probe responded to Cys and emitted strong green fluorescence, whereas it reacted with Hcy/GSH and generated blue fluorescence. Addition of CTAB (cetyl trimethylammonium bromide) was observed to alter the fluorescence color of the reaction product of 4F-2CN and Hcy (from blue to green), but no alteration of the fluorescence color occurred for Cys and GSH. For the very first time, cell imaging experiments showed that the three commonly occurring thiols (Cys/Hcy/GSH) could be differentiated using a single fluorescent probe. In addition, the reaction product of 4F-2CN and Cys exhibits two-photon properties, offering a potentially useful tool for tissue imaging studies. To the best of our knowledge, 4F-2CN is currently the smallest fluorescent probe for thiol detection. We envision that this new and versatile probe will be a useful tool for further elucidating the roles of thiols in biology.

A stimuli-responsive drug delivery system (DDS) with bioactive surface is constructed by end-capping mesoporous silica nanoparticles (MSNs) with functional peptide-coated gold nanoparticles (GNPs). MSNs are first functionalized with acid-labile α-amide-β-carboxyl groups to carry negative charges, and then capped with positively charged GNPs that are decorated with oligo-lysine-containing peptide. The resulting hybrid delivery system exhibits endo/lysosomal pH triggered drug release, and the incorporation of RGD peptide facilitates targeting delivery to αvβ3 integrin overexpressing cancer cells. The system can serve as a platform for preparing diversified multifunctional nanocomposites using various functional inorganic nanoparticles and bioactive peptides.Keywords: charge reversal; controlled release; gold nanoparticles; peptides; targeted delivery

•We reported that a fluorescent probe could detect Cu(II) at subnanomolar concentrations.•Cu(II) is a specific and potent inhibitor of cysteine.•Cu(II) can differentially modulate the reactivity of cysteinyl thiol.•Cell imaging experiments confirmed the inhibitory effects of Cu(II) on Cys's reactivity.In this study, Cu(II)-mediated differential alteration of cysteine (Cys) reactivity is reported by using a Cys-specific fluorescent probe. The probe could react with Cys to give out strong fluorescence. When Cys was preincubated with Cu(II), the fluorescence of the probe was decreased due to the inhibition of Cys's reactivity by Cu(II). Remarkably, experimental results reveal that the probe could detect Cu(II) at subnanomolar concentrations. In contrast, Cu(II) could only partially inhibit the reaction between Cys and Ellman's reagent (DTNB). Furthermore, selectivity experiments show that Cu(II) is a much more potent inhibitor for Cys compared to other metal ions. Cell imaging experiments also confirm the inhibitory effects of Cu(II) on Cys's reactivity in living cells. We envision that the probe could add a useful tool for sensitive and selective detection of Cu(II) for biomedical research.

HNO has recently been found to possess distinct biological functions from NO. Studying the biological functions of HNO calls for the development of sensitive and selective fluorescent probes. Herein, we designed and synthesized a FRET-based ratiometric probe to detect HNO in living cells. Our studies revealed that the probe is capable of detecting HNO in a rapid and ratiometric manner under physiological conditions. In bioimaging studies, the probe displayed a clear color change from blue to green when treated with HNO.Keywords: bioimaging; fluorescent probe; FRET probe; HNO; ratiometric

A new fluorescent probe installed with dual-reactive and dual-quenching groups was rationally designed for highly selective and sensitive sensing of biothiols. The sensitivity of the probe toward thiols was significantly improved by dual-quenching effects. Furthermore the selectivity of the probe was also greatly enhanced by installation of dual-reactive groups.

The development of hydrogels that are responsive to external stimuli in a well-controlled manner is important for numerous biomedical applications. Herein we reported the first example of a hydrogel responsive to hydrogen sulphide (H2S). H2S is an important gasotransmitter whose deregulation has been associated with a number of pathological conditions. Our hydrogel design is based on the functionalization of an ultrashort hydrogelating peptide sequence with an azidobenzyl moiety, which was reported to react with H2S selectively under physiological conditions. The resulting peptide was able to produce hydrogels at a concentration as low as 0.1 wt%. It could then be fully degraded in the presence of excess H2S. We envision that the novel hydrogel developed in this study may provide useful tools for biomedical research.

•A new fluorescent probe based on iminocoumarin benzothiazole was designed to detect H2S.•The probe has high selectively, with 80-fold fluorescence increment upon addition of H2S.•The probe is capable of monitoring the alteration of H2S concentration in living cells.Hydrogen sulfide (H2S) has recently been identified as the third gaseous signaling molecule that is involved in regulating many important cellular processes. We report herein a novel fluorescent probe for detecting H2S based on iminocoumarin benzothiazole scaffold. The probe displayed high sensitivity and around 80-fold increment in fluorescence signal after reacting with H2S under physiological condition. The fluorescent intensity of the probe was linearly related to H2S concentration in the range of 0–100 μM with a detection limit of 0.15 μM (3σ/slope). The probe also showed excellent selectivity towards H2S over other biologically relevant species, including ROS, RSS and RNS. Its selectivity for H2S is 32 folds higher than other reactive sulfur species. Furthermore, the probe has been applied for imaging H2S in living cells. Cell imaging experiments demonstrated that the probe is cell-permeable and can be used to monitor the alteration of H2S concentrations in living cells. We envisage that this probe can provide useful tools to further elucidate the biological roles of H2S.We report a new fluorescent probe based on iminocoumarin benzothiazole to detect H2S selectively, with 80-fold fluorescence increment upon addition of H2S.

Developing robust antibacterial materials is of importance for a wide range of applications such as in biomedical engineering, environment, and water treatment. Herein we report the development of a novel superhydrophobic surface featured with hierarchical architecture and bimetallic composition that exhibits enhanced antibacterial activity. The surface is created using a facile galvanic replacement reaction followed by a simple thermal oxidation process. Interestingly, we show that the surface’s superhydrophobic property naturally allows for a minimal bacterial adhesion in the dry environment, and also can be deactivated in the wet solution to enable the release of biocidal agents. In particular, we demonstrate that the higher solubility nature of the thermal oxides created in the thermal oxidation process, together with the synergistic cooperation of bimetallic composition and hierarchical architecture, allows for the release of metal ions in a sustained and accelerated manner, leading to enhanced antibacterial performance in the wet condition as well. We envision that the ease of fabrication, the versatile functionalities, and the robustness of our surface will make it appealing for broad applications.Keywords: antibacterial; bimetallic; hierarchical; superhydrophobic

By anchoring 1,2,4,5-tetrazine-containing biomolecules onto trans-cyclooctene (TCO)-functionalized slides, a site-specific microarray immobilization approach is described in this study. Compared with existing immobilization methods, our approach offers several distinctive features, including fast kinetics and high chemoselectivity.

A series of new fluorogenic probes for monoamine oxidases (MAOs) were reported based on an oxidation and β-elimination mechanism. The limits of detection of the probes for MAO-A and -B were determined to be 3.5 and 6.0 μg mL−1 respectively. These probes displayed strong activity towards MAOs, especially MAO-B. Cellular imaging studies were also successfully conducted with MCF-7 cells.

Herein we describe a modularly designed colorimetric approach for facile and sensitive detection of proteases. The method employs non-crosslinking AuNP aggregation induced by peptide treated with specific protease. A visible colour change of the AuNPs solution from wine-red to violet-blue is readily observed when the protease-digested peptide is added. This method allows facile and visualized assay of the enzyme activity without any sophisticated instrument, affording both convenience and simplicity. The platform can be readily extended to detect virtually any protease.

We report herein a new site-specific microarray immobilization method based on a biocompatible reaction between terminal cysteine and 2-cyanobenzothiazole (CBT). This immobilization strategy has been successfully applied to anchor small molecules, peptides and proteins onto microarrays.

Proteins are fundamental components of all living systems and critical drivers of biological functions. The large-scale study of proteins, their structures and functions, is defined as proteomics. This systems-wide analysis leads to a more comprehensive view of the intricate signaling transduction pathways that proteins engage in and improves the overall understanding of the complex processes supporting the living systems. Over the last two decades, the development of high-throughput analytical tools, such as microarray technologies, capable of rapidly analyzing thousands of protein-functioning and protein-interacting events, has fueled the growth of this important field. Herein, we review the most recent advancements in microarray technologies, with a special focus on peptide microarray, small molecule microarray, and protein microarray. These technologies have become prominent players in proteomics and have made significant changes to the landscape of life science and biomedical research. We will elaborate on their performance, advantages, challenges, and future directions.

We report herein a fluorescent probe for the detection of hydrogen sulfide (H2S) with high sensitivity and selectivity. The probe is installed with double azide groups and displays high sensitivity to H2S (around 120-fold turn-on response). It reacts with H2S with high selectivity over other reactive sulfur, nitrogen, and oxygen species. The probe was also applied to detect H2S in living cells.We report a new fluorescent probe installed with double azide moieties to detect H2S selectively, with 120-fold fluorescence increment on addition of H2S.

We report herein a novel fluorescent probe based on α,β-unsaturated acyl sulfonamide to detect thiols. The probe has good water solubility and reacts with thiols under aqueous conditions. It reacts selectively with cysteine but not with the other natural amino acids. The probe was subsequently applied to detect intracellular thiols.

By anchoring 1,2,4,5-tetrazine-containing biomolecules onto trans-cyclooctene (TCO)-functionalized slides, a site-specific microarray immobilization approach is described in this study. Compared with existing immobilization methods, our approach offers several distinctive features, including fast kinetics and high chemoselectivity.

A simple molecule, tetrafluoroterephthalonitrile (4F-2CN), was discovered to be an efficient fluorescent probe for detecting biological thiol species. The probe responded to Cys and emitted strong green fluorescence, whereas it reacted with Hcy/GSH and generated blue fluorescence. Addition of CTAB (cetyl trimethylammonium bromide) was observed to alter the fluorescence color of the reaction product of 4F-2CN and Hcy (from blue to green), but no alteration of the fluorescence color occurred for Cys and GSH. For the very first time, cell imaging experiments showed that the three commonly occurring thiols (Cys/Hcy/GSH) could be differentiated using a single fluorescent probe. In addition, the reaction product of 4F-2CN and Cys exhibits two-photon properties, offering a potentially useful tool for tissue imaging studies. To the best of our knowledge, 4F-2CN is currently the smallest fluorescent probe for thiol detection. We envision that this new and versatile probe will be a useful tool for further elucidating the roles of thiols in biology.

A series of new fluorogenic probes for monoamine oxidases (MAOs) were reported based on an oxidation and β-elimination mechanism. The limits of detection of the probes for MAO-A and -B were determined to be 3.5 and 6.0 μg mL−1 respectively. These probes displayed strong activity towards MAOs, especially MAO-B. Cellular imaging studies were also successfully conducted with MCF-7 cells.

We report herein a new site-specific microarray immobilization method based on a biocompatible reaction between terminal cysteine and 2-cyanobenzothiazole (CBT). This immobilization strategy has been successfully applied to anchor small molecules, peptides and proteins onto microarrays.

We report herein a novel fluorescent probe based on α,β-unsaturated acyl sulfonamide to detect thiols. The probe has good water solubility and reacts with thiols under aqueous conditions. It reacts selectively with cysteine but not with the other natural amino acids. The probe was subsequently applied to detect intracellular thiols.

The development of hydrogels that are responsive to external stimuli in a well-controlled manner is important for numerous biomedical applications. Herein we reported the first example of a hydrogel responsive to hydrogen sulphide (H2S). H2S is an important gasotransmitter whose deregulation has been associated with a number of pathological conditions. Our hydrogel design is based on the functionalization of an ultrashort hydrogelating peptide sequence with an azidobenzyl moiety, which was reported to react with H2S selectively under physiological conditions. The resulting peptide was able to produce hydrogels at a concentration as low as 0.1 wt%. It could then be fully degraded in the presence of excess H2S. We envision that the novel hydrogel developed in this study may provide useful tools for biomedical research.

A new fluorescent probe installed with dual-reactive and dual-quenching groups was rationally designed for highly selective and sensitive sensing of biothiols. The sensitivity of the probe toward thiols was significantly improved by dual-quenching effects. Furthermore the selectivity of the probe was also greatly enhanced by installation of dual-reactive groups.

The development of novel antimicrobial materials with high antimicrobial activity and low environmental impact is of importance for global health, but has proven to be challenging. Herein we report a facile mineralization process to create a flower-like, porous antimicrobial agent, which is stable, selective, effective and environmentally benign. This new antimicrobial material is made of organic polydopamine (PD) and inorganic (copper phosphate) components, where the incorporation of PD on the hybrid architecture endows the direct in situ reduction of silver ions into silver nanoparticles (Ag NPs) without the need of external toxic reductants. The combination of Ag NPs and high surface area of the nanoflower results in high selectivity in the antimicrobial activity towards Gram-negative Escherichia coli (E. coli), while leaving co-cultured mammalian cells healthy and intact. Moreover, we show that the hybrid antimicrobial material is stable, and can be easily recovered after use, avoiding the persistent hazard to the environment. We envision that this novel antimicrobial agent may find useful applications for clinical studies and industrial products.