De Novo Green Fluorescent Protein Chromophore-Based Probes for Capturing Latent Fingerprints Using a Portable System.

Rapid visualization of latent fingerprints, preferably at their point of origin, is essential for effective crime scene evaluation. Here, we present a new class of green fluorescent protein chromophore-based fluorescent dyes (LFP-Yellow and LFP-Red) that can be used for real-time visualization of LFPs within 10 s. Compared with traditional chemical reagents for LFPs, these fluorescent dyes are completely water-soluble, exhibit low cytotoxicity, and are harmless to users. Level 1-3 details of the LFPs could be clearly revealed through "off-on" fluorescence signal readout. Additionally, the fluorescent dyes were constructed based on an imidazolinone core and so do not contain pyridine groups or metal ions, which ensures that the DNA is not contaminated during extraction and identification after the LFPs are treated with the dyes. Combined with our as-developed portable system for capturing LFPs, LFP-Yellow and LFP-Red enabled the rapid capture of LFPs. Therefore, these green fluorescent protein chromophore-based probes provide an approach for the rapid identification of individuals who were present at a crime scene.

Cellular Uptake of Cell-Penetrating Peptides Activated by Amphiphilic p-Sulfonatocalix[4]arenes.

We report the synthesis of a series of amphiphilic p-sulfonatocalix[4]arenes with varying alkyl chain lengths (CX4-Cn) and their application as efficient counterion activators for membrane transport of cell-penetrating peptides (CPPs). The enhanced membrane activity is confirmed with the carboxyfluorescein (CF) assay in vesicles and by the direct cytosolic delivery of CPPs into CHO-K1, HCT 116, and KTC-1 cells enabling excellent cellular uptake of the CPPs into two cancer cell lines. Intracellular delivery was confirmed by fluorescence microscopy after CPP entry into live cells mediated by CX4-Cn, which was also quantified after cell lysis by fluorescence spectroscopy. The results present the first systematic exploration of structure-activity relationships for calixarene-based counterion activators and show that CX4-Cn are exceptionally effective in cellular delivery of CPPs. The dodecyl derivative, CX4-C12, serves as best activator. A first mechanistic insight is provided by efficient CPP uptake at 4 °C and in the presence of the endocytosis inhibitor dynasore, which indicates a direct translocation of the CPP-counterion complexes into the cytosol and highlights the potential benefits of CX4-Cn for efficient and direct translocation of CPPs and CPP-conjugated cargo molecules into the cytosol of live cells.

Chemoselective Fluorogenic Bioconjugation of Vicinal Dithiol-Containing Proteins for Live Cellular Imaging via Small Molecular Conjugate Acceptors.

To develop small molecular fluorogenic tools for the chemoselective labeling of vicinal dithiol-containing proteins (VDPs) in live cells is important for studying intracellular redox homeostasis. With this research, we developed small molecule-based fluorescent probes, achieving selective labeling of VDPs through thiol-thiol substitutions on bisvinylogous thioester conjugated acceptors (IDAs). Initially, IDAs demonstrated its ability to bridge vicinal cysteine-sulfhydryls on a peptide as a mimic. Then, the peptide complex could be decoupled to recover the original peptide-SH in the presence of dithiothreitol. Furthermore, fluorometric signal amplification of the fluorescent probes occurred with high sensitivity, low limit of detection, and selectivity toward vicinal dithiols on reduced bovine serum albumin, as an example of real world VDPs. More importantly, the probes were utilized successfully for labeling of endogenous VDPs at different redox states in live cells. Thus, the bisvinylogous thioester-based receptor as a functional probe represents a new platform for uncovering the function of VDPs in live cells.

Dual-Channel Fluorescent Probe for the Simultaneous Monitoring of Peroxynitrite and Adenosine-5'-triphosphate in Cellular Applications.

Changes in adenosine triphosphate (ATP) and peroxynitrite (ONOO-) concentrations have been correlated in a number of diseases including ischemia-reperfusion injury and drug-induced liver injury. Herein, we report the development of a fluorescent probe ATP-LW, which enables the simultaneous detection of ONOO- and ATP. ONOO- selectively oxidizes the boronate pinacol ester of ATP-LW to afford the fluorescent 4-hydroxy-1,8-naphthalimide product NA-OH (λex = 450 nm, λem = 562 nm or λex = 488 nm, λem = 568 nm). In contrast, the binding of ATP to ATP-LW induces the spirolactam ring opening of rhodamine to afford a highly emissive product (λex = 520 nm, λem = 587 nm). Due to the differences in emission between the ONOO- and ATP products, ATP-LW allows ONOO- levels to be monitored in the green channel (λex = 488 nm, λem = 500-575 nm) and ATP concentrations in the red channel (λex = 514 nm, λem = 575-650 nm). The use of ATP-LW as a combined ONOO- and ATP probe was demonstrated using hepatocytes (HL-7702 cells) in cellular imaging experiments. Treatment of HL-7702 cells with oligomycin A (an inhibitor of ATP synthase) resulted in a reduction of signal intensity in the red channel and an increase in that of the green channel as expected for a reduction in ATP concentrations. Similar fluorescence changes were seen in the presence of SIN-1 (an exogenous ONOO- donor).

Förster resonance energy transfer (FRET)-based small-molecule sensors and imaging agents.

In this tutorial review, we will explore recent advances in the construction and application of Förster resonance energy transfer (FRET)-based small-molecule fluorescent probes. The advantages of FRET-based fluorescent probes include: a large Stokes shift, ratiometric sensing and dual/multi-analyte responsive systems. We discuss the underlying energy donor-acceptor dye combinations and emphasise their applications for the detection or imaging of cations, anions, small neutral molecules, biomacromolecules, cellular microenvionments and dual/multi-analyte responsive systems.

A new long-wavelength fluorescent probe for tracking peroxynitrite in live cells and inflammatory sites of zebrafish.

Peroxynitrite (OONO-), as a reactive oxygen species (ROS), would be mostly profoundly implicated in diseases such as inflammation in organisms. However, bioimaging of ONOO- still faces difficulties owing to the shortage of bioimaging and real-time dynamic tracking distribution of ROS in inflammation. To address this challenge, we designed and synthesized a long-wavelength fluorescent probe based on tricyanofuran (ACDM-BE), which exhibits a fast response (response time is 40 s), high selectivity and great sensitivity (LOD is approximately 21 nM) towards ONOO-. ACDM-BE was shown to be capable of detecting ONOO- in living cells and monitor the changes in ONOO- levels under the stimulus of various concentrations of SIN-1 (from 100 to 700 µM), which was successfully tracked by the fluorescence changes in live cells. It is worth noting that ACDM-BE further demonstrated its ability to track the dynamic changes of the level of ONOO- in the inflammatory sites of larval zebrafish. Thus, ACDM-BE could be employed as an efficient tool for exploiting the role of ONOO- in inflammation in living biosystems.

A renewable test strip combined with solid-state ratiometric fluorescence emission spectra for the highly selective and fast determination of hydrazine gas.

N2H4 is one of the most toxic and explosive chemicals. In particular, N2H4 vapor is dangerously unstable. However, the determination of N2H4 gas has lagged behind the detection of N2H4 in aqueous solution or biological fluid in terms of the crucial ability to discriminate and quantify N2H4. Despite the multitude of fluorescent probe-coated test strips that have been used for N2H4 gas detection, these techniques mainly rely on the observation by eye of fluorescence color changes, which will hamper the discrimination of subtle color changes on the test strips due to the limited capacity of the eye to perceive and differentiate colors. Herein, we developed a new technique combining a renewable test strip and solid-state ratiometric fluorescence readout. The results confirmed that the prepared test strips could be used as a solid-state fluorescence sensor for the fast capture (within 5 min) and quantification of N2H4 gas. Additionally, the test strips could be easily renewable. Thus, this test strip-based approach provides an efficient tool for N2H4 gas detection, which is particularly significant for the real-time monitoring of N2H4 gas in airports and the environmental atmosphere in chemical plant regions. This design concept presents a new avenue for developing solid-state fluorescence sensors for gas detection.

3D carbon nanosphere and gold nanoparticle-based voltammetric cytosensor for cell line A549 and for early diagnosis of non-small cell lung cancer cells.

An electrochemical cytosensor for the detection of the non-small-cell lung cancer cell line A549 (NSCLC) had been developed. A microwave-hydrothermal method was employed to prepare monodisperse colloidal carbon nanospheres (CNSs). Gold nanoparticles (AuNPs) were placed on the surface of the colloidal CNSs by self-assembly to obtain 3D-structured microspheres of the type CNS@AuNP. The results of an MTT assay show the microspheres to possess good biocompatibility. The CNS@AuNP nanocomposite was then placed, in a chitosan film, on a glassy carbon electrode (GCE). The voltammetric signals and detection sensitivity are significantly enhanced owing to the synergistic effect of CNSs and AuNPs. A cytosensor was then obtained by immobilization of antibody against the carcinoembryonic antigen (which is a biomarker for NSCLC) on the GCE via crosslinking with glutaraldehyde. Hexacyanoferrate is used as an electrochemical probe, and the typical working voltage is 0.2 V (vs. SCE). If exposed to A549 cells, the differential pulse voltammetric signal decreases in the 4.2 × 10-1 to 4.2 × 10-6 cells mL-1 concentration range, and the detection limit is 14 cells mL-1 (at S/N = 3). Graphical abstract Schematic presentation of design strategy and fabrication process of the electrochemical cytosensor for A549 cells. (CNS: carbon nanospheres; GA: glutaraldehyde; PEI: polyethyleneimine; AuNPs: gold nanoparticles; BSA: Bovine serum albumin).

Hyaluronic acid-grafted three-dimensional MWCNT array as biosensing interface for chronocoulometric detection and fluorometric imaging of CD44-overexpressing cancer cells.

A sandwich-type electrochemical cytosensor is described for quantitative determination of CD44-overexpressing HeLa cells. Hyaluronic acid (HA) acts as a targeting molecule that was in-situ incorporated into the sensor based on the use of an indium tin oxide (ITO) electrode modified with multi-walled carbon nanotubes (MWCNTs). The 3D-MWCNT structure is shown to strongly improve the electronic properties and surface chemical reactivities. The HA-modified sensor exhibits a highly sensitive response to HeLa cells. A sandwiched hybridization protocol was then established using BIO [an N-butyl-4-(6'-aminohexyl)amino-1,8-naphthalimide probe modified with HA] as the tracing labels of the fluorescent probes for targeting CD44-positive tumor cells. The signal amplification was thereby maximized and measured by chronocoulometry. The binding of CD44-positive HeLa cells to the HA modified sensing layer causes a decrease in chronocoulometric response. The signal decreases linearly in the 2.1 × 102 to 2.1 × 107 HeLa cells·mL-1 concentration range with a detection limit of 70 cells·mL-1. Such a sandwich-type assay may be tailored as a sensitive candidate for detecting low levels of tumor cells. Graphical abstract Schematic of a sandwich cytosensor based on hyaluronic acid-grafted 3D-MWCNT as biosensing interface and BIO as fluorescent probe. This biosensor possessed excellent electrochemical activity, high sensitivity and selectivity, providing a dynamical tracking and detecting platform for CD44-positive tumor cells.

An ultrasensitive multi-walled carbon nanotube-platinum-luminol nanocomposite-based electrochemiluminescence immunosensor.

An ultrasensitive electrochemiluminescence (ECL) immunosensor for carbohydrate antigen 19-9 (CA19-9) detection using multi-walled carbon nanotube-platinum-luminol nanocomposites (MWCNT-Pt-luminol) as nanointerface and signal tags was designed. First, the MWCNT-Pt-luminol nanocomposite was decorated on the surface of a glassy carbon electrode (GCE) through the film-forming properties of chitosan (Chi). Then, the CA19-9 antibody (anti-CA19-9) was attached to the modified electrode surface via crosslinking with glutaraldehyde (GA). When CA19-9 was captured by its antibody immobilized on the immune platform via immunoreaction, the ECL signal intensity decreased. Under optimal conditions, the proposed ECL immunosensor showed excellent performance for CA19-9 ranging from 0.0001 U mL-1 to 10.0 U mL-1, with a detection limit of 0.000046 U mL-1 (S/N = 3) and a correlation coefficient of R = 0.9980. This is attributed to the fact that the MWCNTs-Pt nanomaterial has excellent conductivity and it could facilitate the decomposition of H2O2 to generate various reactive oxygen species (ROSs); thus, the ECL signals of luminol were effectively amplified and the sensitivity of the sensor was greatly increased. The prepared ECL immunosensor displayed simple, fast analysis, excellent stability, good reproducibility, and high specificity. Moreover, the developed ECL immunosensor provided satisfactory results in the determination of CA19-9 in real human serum samples.

Dual-Modal Colorimetric/Fluorescence Molecular Probe for Ratiometric Sensing of pH and Its Application.

As traditional pH meters cannot work well for minute regions (such as subcellular organelles) and in harsh media, molecular pH-sensitive devices for monitoring pH changes in diverse local heterogeneous environments are urgently needed. Here, we report a new dual-modal colorimetric/fluorescence merocyanine-based molecular probe (CPH) for ratiometric sensing of pH. Compared with previously reported pH probes, CPH bearing the benzyl group at the nitrogen position of the indolium group and the phenol, which is used as the acceptor for proton, could respond to pH changes immediately through both the ratiometric fluorescence signal readout and naked-eye colorimetric observation. The sensing process was highly stable and reversible. Most importantly, the suitable pKa value (6.44) allows CPH to presumably accumulate in lysosomes and become a lysosome-target fluorescent probe. By using CPH, the intralysosomal pH fluctuation stimulated by antimalaria drug chloroquine was successfully tracked in live cells through the ratiometric fluorescence images. Additionally, CPH could be immobilized on test papers, which exhibited a rapid and reversible colorimetric response to acid/base vapor through the naked-eye colorimetric analysis. This proof-of-concept study presents the potential application of CPH as a molecular tool for monitoring intralysosomal pH fluctuation in live cells, as well as paves the way for developing the economic, reusable, and fast-response optical pH meters for colorimetric sensing acid/base vapor with direct naked-eye observation.

A Functional CT Contrast Agent for In Vivo Imaging of Tumor Hypoxia.

Hypoxia, which has been well established as a key feature of the tumor microenvironment, significantly influences tumor behavior and treatment response. Therefore, imaging for tumor hypoxia in vivo is warranted. Although some imaging modalities for detecting tumor hypoxia have been developed, such as magnetic resonance imaging, positron emission tomography, and optical imaging, these technologies still have their own specific limitations. As computed tomography (CT) is one of the most useful imaging tools in terms of availability, efficiency, and convenience, the feasibility of using a hypoxia-sensitive nanoprobe (Au@BSA-NHA) for CT imaging of tumor hypoxia is investigated, with emphasis on identifying different levels of hypoxia in two xenografts. The nanoprobe is composed of Au nanoparticles and nitroimidazole moiety which can be electively reduced by nitroreductase under hypoxic condition. In vitro, Au@BSA-NHA attain the higher cellular uptake under hypoxic condition. Attractively, after in vivo administration, Au@BSA-NHA can not only monitor the tumor hypoxic environment with CT enhancement but also detect the hypoxic status by the degree of enhancement in two xenograft tumors with different hypoxic levels. The results demonstrate that Au@BSA-NHA may potentially be used as a sensitive CT imaging agent for detecting tumor hypoxia.

Multiwalled carbon nanotubes/gold nanocomposites-based electrochemiluminescent sensor for sensitive determination of bisphenol A.

An electrochemiluminescence (ECL) sensor for bisphenol A was proposed by using L-cysteine-functionalized multiwalled carbon nanotubes/gold nanocomposites-modified glassy carbon electrode (MWCNTs-Au/GCE) based on ECL of peroxydisulfate solution. The ECL behaviors of peroxydisulfate solution had been investigated at the chitosan/MWCNTs-Au/GCE, and bisphenol A was found to have quenching effects on the ECL of peroxydisulfate solution. Both Au nanoparticles (AuNPs) and multiwalled CNTs could promote the electron transfer and synergetically amplify the ECL signal of peroxydisulfate solution. Under the optimized conditions, the ECL signal intensity was linear with the concentration of bisphenol A in the concentration range between 0.25 and 100 µM (R = 0.9931) with a detection limit (S/N = 3) of 0.083 µM. The constructed ECL sensor has the advantages of simplicity, sensitivity, good selectivity, and reproducibility, exhibiting a great potential application in the determination of bisphenol A.

Selective and ratiometric fluorescent trapping and quantification of protein vicinal dithiols and in situ dynamic tracing in living cells.

Protein vicinal dithiols play fundamental roles in intracellular redox homeostasis due to their involvement in protein synthesis and function through the reversible vicinal dithiol oxidation to disulfide. To provide quantitative information about the global distribution and dynamic changes of protein vicinal dithiols in living cells, we have designed and synthesized a ratiometric fluorescent probe (VTAF) for trapping of vicinal dithiol-containing proteins (VDPs) in living cells. VTAF exhibits a ratiometric fluorescence signal upon single excitation, which enables self-calibration of the fluorescence signal and quantification of endogenous vicinal dithiols of VDPs. Its potential for in situ dynamic tracing of changes of protein vicinal dithiols under different cellular redox conditions was exemplified. VTAF facilitated the direct observation of subcellular distribution of endogenous VDPs via ratiometric fluorescence imaging and colocalization assay. And the results suggested that there are abundant VDPs in mitochondria. Moreover, some redox-sensitive VDPs are also present on cell surface which can respond to redox stimulus. This ratiometric fluorescence technique presents an important extension to previous fluorescence intensity-based probes for trapping and quantifying protein vicinal dithiols in living cells, as well as its visible dynamic tracing of VDPs.

Unique tri-output optical probe for specific and ultrasensitive detection of hydrazine.

An optical probe based on colorimetric and ratiometric as well as chemiluminometric signal outputs is developed for the specific detection of hydrazine. On the basis of a Gabriel-type reaction, hydrazinolysis of a simple probe CF (4-phtalamide-N-(4'-methylcoumarin) naphthalimide) produces both the fluorescence of 7-amino-4-methylcoumarin with the max emission wavelength changed from 480 to 420 nm (along with a color change from yellow to transparent) and the luminol chemiluminescence activated by H2O2 with a max emission wavelength at 450 nm. The experimental detection limit of hydrazine is 3.2 ppb (0.1 µM). Selectivity experiments proved CF has excellent selectivity to hydrazine over other interfering substances. Probe CF was also successfully applied in the vapor hydrazine detection over other interfering volatile analytes. Furthermore, the probe CF loaded thin-layer chromatography (TLC) plate for vapor hydrazine detection limit is 5.4 mg/m(3) which is well below the half lethal dose of hydrazine gas for mice (LC50(mice), 330 mg/m(3)) and National Institute of Occupational Safety and Health's immediately dangerous to life or health limit (NIOSHIDLH, 66 mg/m(3)). With H2O2, only hydrazinolysis product luminol can be lighted at 450 nm, other species have no signal. Probe CF can also be used for the detection of hydrazine in HeLa cells.

A colloidal gold nanoparticle-based immunochromatographic test strip for rapid and convenient detection of Staphylococcus aureus.

An immunochromatographic test strip using gold nanoparticles-staphylococcus aureus monoclonal antibody conjugates was developed for the rapid and convenient detection of staphylococcus aureus based on a double-antibody sandwich format. The detection limit and the detection rate of this test strip is 10(3) CFU /mL and 98.7%, respectively. It could be used for the rapid detection of staphylococcus aureus in food and the results can be visually identified by the naked eye within 10 min. Compared with conventional bacterial detection methods, this developed immunochromatographic assay based test strip has several advantages including simple, fast, low-cost, favorable sensitivity and specificity, exhibiting a great potential for application in food safety control systems and clinical diagnosis.

Versatile probes for the selective detection of vicinal-dithiol-containing proteins: design, synthesis, and application in living cells.

Endogenous vicinal-dithiol-containing proteins (VDPs) that have two thiol groups close to each other in space play a significant importance in maintaining the cellular redox microenvironment. Approaches to identify VDPs mainly rely on monitoring the different concentration of monothiol and total thiol groups or on indirect labeling of vicinal thiols by using p-aminophenylarsenoxide (PAO). Our previous work has reported the direct labeling of VDPs with a highly selective receptor PAO analogue, which could realize fluorescence detection of VDPs directly in living cells. Herein, we developed a conjugated approach to expand detectable tags to nitrobenzoxadiazole (NBD), fluorescein, naphthalimide, and biotin for the synthesis of a series of probes. Different linkers have also been introduced toward conjugation of VTA2 with these functional tags. These synthesized flexible probes with various features will offer new tools for the potential identification and visualization of vicinal dithiols existing in different regions of VDPs in living cells. These probes are convenient tools for proteomics studies of various disease-related VDPs and for the discovery of new drug targets.

In situ visualization and detection of protein sulfenylation responses in living cells through a dimedone-based fluorescent probe.

Sulfenylation is one of the reversible post-translational modifications, playing significant roles in cellular redox homeostasis and signaling systems. Herein, small fluorescent probe (CPD and CPDDM) based live-cell labelling technology for the visualization of protein sulfenylation responses in living cells has been developed. This approach enables the detection of protein sulfenylation without the need for cell lysis, fixation or purification, and permits the noninvasive study of protein sulfenylation in live cells through the direct fluorescent readout. This technology also can realize dynamic tracking of protein sulfenylation in situ with minimal perturbation to sulfenylated proteins and less interference with cellular function. Information on the global distribution and dynamic changes of endogenous protein sulfenylation has been obtained.

In2O3/Bi2S3 S-scheme Heterojunction-Driven Molecularly Imprinted Photoelectrochemical Sensor for Ultrasensitive Detection of Florfenicol.

Florfenicol (FF) raises significant human health and environmental concerns due to its toxicity to the hematology system and the potential spread of antibiotic-resistant genes. Here, a highly sensitive molecularly imprinted photoelectrochemical (PEC) sensor, featuring an In2O3/Bi2S3 S-scheme heterojunction, is proposed to detect FF without an external voltage supply. Compared with conventional II-type heterojunctions, S-scheme heterojunctions efficiently promote carrier separation and enhance the redox capability of the photocatalytic system. This allows more dissolved O2 and H2O molecules to participate in the redox reaction, resulting in an amplified and stabilized photocurrent response. The electron transfer in the S-scheme heterojunction is confirmed via electron spin resonance (ESR). With the molecular imprinting technique, this PEC platform exhibits exceptional selectivity, wide linear range (1.0 × 10-4-1.0 × 104 ng mL-1), low detection limit (6.4 × 10-5 ng mL-1), and applicability in real milk and chicken samples. This work not only showcases a PEC platform for accurately and portably detecting drugs but also proposes a viable approach for designing S-scheme heterojunctions in sensing analysis.

[Chemical approaches for trapping protein thiols and their oxidative modification].

Redox signal transduction, especially the oxidative modification of proein thiols, correlates with many diseases and becomes an expanding research area. However, there was rare method for quick and specific detection of protein thiols and their oxidative modification in living cells. In this article, we review the current chemical strategies for the detection and quantification of protein thiols and related cysteine oxidation. We also look into the future of the development of fluorescent probes for protein thiols and their potential application in the research of reactive cysteine proteomes and early detection of redox-related diseases.