Coralyne cation, a fluorescent probe for general detection in planar chromatography

A large number of analytes, including non-fluorescent ones, can be sensitively detected by fluorescence scanning densitometry using silica gel HPTLC plates impregnated with a solution of coralyne cation. This is carried out by the variation, increase or decrease, that the corresponding analyte induces on native coralyne emission at a given excitation wavelength. A similar phenomenon was previously described for berberine cation, and Reichardt's dye probes. However, the sensitivity of coralyne in HPTLC detection of non-fluorescent, structurally different analytes (e.g., long-chain alkanes, alcohols, alkylbromides, neutral lipids) is superior to that of the above-mentioned probes. In this work, the analytical viability of this phenomenon for HPTLC detection using coralyne as a probe is explored, and fluorescent responses of a number of analytes on the coralyne system are rationalized in the light of a previously proposed model. This establishes that the resulting intensity for a probe in the presence of a given compound can be explained as a balance between radiative (contribution of non-specific interactions) and non-radiative processes (specific interactions), the latter producing fluorescence quenching. Experimental results and proposed model suggest that this phenomenon may be general for practically all kinds of analytes.     

Photocatalytically Powered Matchlike Nanomotor for Light‐Guided Active SERS Sensing

Surface enhanced Raman spectroscopy (SERS) is a powerful optical sensing technique that can detect analytes of extremely low concentrations. However, the presence of enough SERS probes in the detection area and a close contact between analytes and SERS probes are critical for efficient acquisition of a SERS signal. Presented here is a light‐powered micro/nanomotor (MNM) that can serve as an active SERS probe. The matchlike AgNW@SiO₂ core–shell structure of the nanomotors work as SERS probes based on the shell‐isolated enhanced Raman mechanism. The AgCl tail serves as photocatalytic nanoengine, providing a self‐propulsion force by light‐induced self‐diffusiophoresis. The phototactic behavior was utilized to achieve enrichment of the nanomotor‐based SERS probes for on‐demand biochemical sensing. The results demonstrate the possibility of using photocatalytic nanomotors as active SERS probes for remote, light‐controlled, and smart biochemical sensing on the micro/nanoscale.     

"Turn-on" fluorescent sensing with "reactive" probes

Chemical probes are valuable tools for the investigation of biochemical processes, diagnosis of disease markers, detection of hazardous compounds, and other purposes. Therefore, the development of chemical probes continues to grow through various approaches with different disciplines and design strategies. Fluorescent probes have received much attention because they are sensitive and easy-to-operate, in general. To realize desired selectivity toward a given analyte, the recognition site of a fluorescent probe is designed in such a way to maximize the binding interactions, usually through weak molecular forces such as hydrogen bonding, toward the analyte over other competing ones. In addition to such a supramolecular approach, the development of fluorescent probes that sense analytes through chemical reactions has witnessed its usefulness for achieving high selectivity, in many cases, superior to that obtainable by the supramolecular approach. Creative incorporations of the reactive groups to latent fluorophores have provided novel chemical probes for various analytes. In this feature article, we overview the recent progress in the development of turn-on fluorescent probes that are operating through chemical reactions triggered by target analytes. Various chemical reactions have been implemented in the development of many reactive probes with very high selectivity and sensitivity toward target analytes. A major emphasis has been focused on the type of chemical reactions utilized, with the hope that further explorations can be made with new chemical reactions to develop reactive probes useful for various applications.     

A general strategy to quantify analytes through fluorescence chromaticity and luminosity

To realize a fast, easy-operation and precise way using fluorescence probes to quantify analytes is a goal to facilitate detection, especially in situ. Herein, we are reporting an approach which can be generally employed for the differentiation and quantitation of analytes through fluorescence chromaticity and luminosity. Seven representative fluorescent probes, targeting pH, cysteine, hydrogen sulfide, hydrogen peroxide, palladium and hydrazine, were synthesized and tested. Without utilizing costly instrumentations, portable devices were employed to collect data of photographs from the fluorescence samples in responses to different analytes. Subsequently, the photographic images were digitally processed to generate calibration curves between chromaticity/luminosity verse concentrations after mapping to the CIE 1931 xyY standard color space. Good linear calibration curves and quantitative analysis of unknown samples with low errors through the spectral technology demonstrated the reliability of this method. Thus, we showed the analytical method with a simple and on-site constructible/portable device which is promising for applications in more fluorescence probes.     

A general strategy to quantify analytes through fluorescence chromaticity and luminosity

To realize a fast, easy-operation and precise way using fluorescence probes to quantify analytes is a goal to facilitate detection, especially in situ. Herein, we are reporting an approach which can be generally employed for the differentiation and quantitation of analytes through fluorescence chromaticity and luminosity. Seven representative fluorescent probes, targeting pH, cysteine, hydrogen sulfide, hydrogen peroxide, palladium and hydrazine, were synthesized and tested. Without utilizing costly instrumentations, portable devices were employed to collect data of photographs from the fluorescence samples in responses to different analytes. Subsequently, the photographic images were digitally processed to generate calibration curves between chromaticity/luminosity verse concentrations after mapping to the CIE 1931 xyY standard color space. Good linear calibration curves and quantitative analysis of unknown samples with low errors through the spectral technology demonstrated the reliability of this method. Thus, we showed the analytical method with a simple and on-site constructible/portable device which is promising for applications in more fluorescence probes     

Development of three end-capped para-benzoyl calix[4,6, or 8]arene bonded stationary phases for HPLC

Three end-capped para-benzoyl calixarene bonded silica gel stationary phases are prepared and characterized by elemental analysis, infrared spectroscopy, and thermal analysis. The comparison and selectivity of these phases are investigated by using PAHs, disubstituted benezene, and naphthalene positional isomers as probes. Possible separation mechanism based on the different interactions between calixarenes and analytes are discussed. The results indicate that the separation for those analytes are influenced by the supramolecular interaction including π-π interaction, π-electron transfer interactions, space steric hindrance, and hydrogen bonding interaction on the calixarene columns. Importantly, the aromatic probes with polar groups such as -OH, -NO(2), and -NH(2) could regulate the selectivity of calixarene-bonded stationary phases.     

Ionization of Volatile Organics and Nonvolatile Biomolecules Directly from a Titanium Slab for Mass Spectrometric Analysis

Atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) and electrospray ionization (ESI)-MS can cover the analysis of analytes from low to high polarities. Thus, an ion source that possesses these two ionization functions is useful. Atmospheric surface-assisted ionization (ASAI), which can be used to ionize polar and nonpolar analytes in vapor, liquid, and solid forms, was demonstrated in this study. The ionization of analytes through APCI or ESI was induced from the surface of a metal substrate such as a titanium slab. ASAI is a contactless approach operated at atmospheric pressure. No electric contacts nor any voltages were required to be applied on the metal substrate during ionization. When placing samples with high vapor pressure in condensed phase underneath a titanium slab close to the inlet of the mass spectrometer, analytes can be readily ionized and detected by the mass spectrometer. Furthermore, a sample droplet (~2 μL) containing high-polarity analytes, including polar organics and biomolecules, was ionized using the titanium slab. One titanium slab is sufficient to induce the ionization of analytes occurring in front of a mass spectrometer applied with a high voltage. Moreover, this ionization method can be used to detect high volatile or polar analytes through APCI-like or ESI-like processes, respectively.     

Nucleic acid-based fluorescent probes and their analytical potential

It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays.     

Design and synthesis of labeling reagents (MS probes) for highly sensitive electrospray ionization mass spectrometry and their application to the detection of carbonyl, alcohol, carboxylic acid and primary amine samples.

Novel labeling reagents, called MS probes, which possess a positively charged quaternary amine moiety and can transform a neutral analyte into a charged compound by simply mixing with the analyte and allowing the mixture to stand from several minutes to 30 min at room temperature or while heating to 50 degrees C, were designed and synthesized for the highly sensitive detection of carbonyl, alcohol, carboxylic acid and primary amine samples by electrospray ionization mass spectrometry (ESI-MS). The positively charged products can be detected with high sensitivity in an ESI-MS system, which is the most popular liquid MS instrument. All of the labeled products showed a remarkably large increase in the molecular-ion peak abundance detection sensitivity of over 500-fold at picomolar concentration levels compared to that of unlabeled analytes in an ESI-MS system. These MS probes, used together with liquid MS detection, are widely applicable as a convenient method for the highly sensitive detection of less than picomolar levels of analytes, and therefore greatly enhance the power of ESI-MS analysis.     

Application of self-organizing maps for the classification of chromatographic systems and prediction of values of chromatographic quantities

The separation of a complex mixture of inorganic and organic anions by ion chromatography–capillary electrophoresis using a cationic polymer added to the background electrolyte and indirect UV detection has been studied. The addition of unmodified polymer to an electrolyte suitable for indirect detection resulted in the appearance of a system peak due to the counter-anion on the polymer and while the position of the analytes relative to this system peak could be changed, this was found to be an unacceptable approach for mixtures of large numbers of analytes. Although conversion of the polymer to replace the counter-ion with the indirect UV detection probe ion simplified the system, this approach restricted the flexibility of the system because the probe and polymer concentration were necessarily linked. This limitation could be overcome by selecting the appropriate type of probe ion, with probes having a low ion-exchange selectivity coefficient providing greater retention of analytes than probes with a high ion-exchange selectivity coefficient. Three electrolyte systems with different probes (benzoate, chromate and phthalate) were modelled using a previously derived migration equation and this was used to optimise the electrolyte composition to enable the separation of a mixture of 24 inorganic and organic anions within 7 min. The electrolyte composition was then optimised for the analysis of anions in Bayer liquor with the final separation selectivity being substantially improved for selected key analytes.     

Carbon nanotubes as affinity probes for peptides and proteins in MALDI MS analysis

Recently, carbon nanotubes (CNTs) have been reported to be an effective MALDI matrix for small molecules (Anal. Chem.2003, 75, 6191). In a somewhat related study, we have employed CNTs produced by using NaH-treated anodic aluminum oxide (Na@AAO) as a reactive template as the assisting matrix for MALDI analysis upon the addition of high concentrations of citrate buffer. Our results indicate that the mass range can be extended to ca. 12,000 Da and that alkali metal adducts of analytes are effectively reduced. Furthermore, we have employed citric acid-treated CNTs as affinity probes to selectively concentrate traces of analytes from aqueous solutions. High concentrations of salts and surfactants in the sample solutions are also tolerated. This approach is very suitable for the MALDI analysis of small proteins, peptides, and protein enzymatic digest products.     

基于三苯胺的荧光探针设计、合成与应用研究进展

螺旋桨结构的三苯胺荧光团既能作为强的电子供体,又能作为潜在的聚集诱导发光(AIE)骨架.同时,三苯胺衍生物很容易通过简单的反应进行结构修饰,如醛基、氨基、硼酸基、卤素、乙炔基等取代的三苯胺能够发生缩合反应或偶联反应等,进一步功能化.因此,功能性三苯胺类化合物被广泛用于太阳能电池、荧光染料、固态发光材料和荧光探针的分子设计中.根据三苯胺基荧光探针的检测对象,将其分为阳离子、阴离子和中性小分子荧光探针三类,并从分子的结构和性能出发,重点综述了近五年来国内外三苯胺基荧光探针在分子设计、合成与检测应用方面的最新进展.展望未来,构建近红外发光和高量子效率的AIE荧光探针值得关注.     

A UPLC-MS/MS assay of the "Pittsburgh cocktail": six CYP probe-drug/metabolites from human plasma and urine using stable isotope dilution

The efficiency of drug metabolism by a single enzyme can be measured as the fractional metabolic clearance which can be used as a measure of whole body activity for that enzyme. Measurement of activity of multiple enzymes simultaneously is feasible using a cocktail approach, however, analytical approach using different assays for drug probes can be cumbersome. A quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based method for the rapid measurement of six cytochrome P450 (CYP) probe drugs and their relevant metabolites is described. The six specific probe substrates/metabolites are caffeine/paraxanthine (CYP1A2), flurbiprofen/4'-hydroxyflurbiprofen (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), debrisoquine/4-hydroxydebrisoquine (CYP2D6), chlorzoxazone/6'-hydroxychlorzoxazone (CYP2E1) and dapsone/N-monoacetyldapsone (NAT2). These probes were quantified by stable isotope dilution from plasma and urine. The present workflow provides a robust, fast and sensitive assay for the "Pittsburgh cocktail", and has been successfully applied to a clinical phenotyping study of liver disease. A representative group of 17 controls and patients with chronic liver disease were administered orally caffeine (100 mg), chlorzoxazone (250 mg), debrisoquine (10 mg), mephenytoin (100 mg), flurbiprofen (50 mg) and dapsone (100 mg). Urine (0 through 8 h) and plasma (4 and 8 h) samples were analyzed for drug/metabolite amounts by stable isotope dilution UPLC-MS/MS. The phenotypic activity of drug metabolizing enzymes was investigated with 17 patient samples. Selected reaction monitoring (SRM) was optimized for each drug and metabolite. In the method developed, analytes were resolved by reversed-phase by development of a gradient using a water/methanol solvent system. SRM of each analyte was performed in duplicate on a triple quadrupole mass spectrometer utilizing an 8 min analytical method each, one with the source operating in the positive mode and one in the negative mode, using the same solvent system. This method enabled quantification of each drug (caffeine, chlorzoxazone, debrisoquine, mephenytoin, flurbiprofen, and dapsone) and its resulting primary metabolite in urine or plasma in patient samples. The method developed and the data herein demonstrate a robust quantitative assay to examine changes in CYP enzymes both independently or as part of a cocktail. The clinical use of a combination of probe drugs with UPLC-MS/MS is a highly efficient tool for the assessment of CYP enzyme activity in liver disease.     

Surface modified silver selinide nanoparticles as extracting probes to improve peptide/protein detection via nanoparticles-based liquid phase microextraction coupled with MALDI mass spectrometry

We report the first use of functionalized Ag₂Se nanoparticles (NPs) as effective extracting probes for NPs-based liquid-phase microextraction (NPs-LPME) to analyze hydrophobic peptides and proteins from biological samples (urine and plasma) and soybean in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Surface modified functional groups such as octadecanethiol (ODT) and 11-mercaptoundecanoic acid (MUA) on Ag₂Se NPs were found to play an important role for efficient extraction of peptides and proteins from test samples through hydrophobic interactions. The peptides can be efficiently extracted using functionalized Ag₂Se NPs as extracting probes in the presence of high concentration of matrix interferences such as 4 M urea, 0.5% Triton X-100 and 3% NaCl. Ag₂Se@ODT NPs have shown better extraction efficiency and detection sensitivity for peptides than Ag₂Se@MUA NPs, bare Ag₂Se NPs and conventional MALDI-MS. The LODs are 20-68 nM for valinomycin and 100-180 nM for gramicidin D using Ag₂Se@ODT NPs-LPME in the MALDI-MS. The current approach is highly sensitive and the target analytes can be effectively isolated without sample loss and efficiently analyzed in MALDI-MS.     

Fluorescent probes for detection of biothiols based on “aromatic nucleophilic substitution-rearrangement” mechanism

“Aromatic nucleophilic substitution-rearrangement (SNAr-rearrangement)” mechanism provided a powerful tool to design fluorescent probes for the discrimination between biothiols.     

Identification of Epoxide Functionalities in Protonated Monofunctional Analytes by Using Ion/Molecule Reactions and Collision-Activated Dissociation in Different Ion Trap Tandem Mass Spectrometers

A mass spectrometric method has been delineated for the identification of the epoxide functionalities in unknown monofunctional analytes. This method utilizes gas-phase ion/molecule reactions of protonated analytes with neutral trimethyl borate (TMB) followed by collision-activated dissociation (CAD) in an ion trapping mass spectrometer (tested for a Fourier-transform ion cyclotron resonance and a linear quadrupole ion trap). The ion/molecule reaction involves proton transfer from the protonated analyte to TMB, followed by addition of the analyte to TMB and elimination of methanol. Based on literature, this reaction allows the general identification of oxygen-containing analytes. Vinyl and phenyl epoxides can be differentiated from other oxygen-containing analytes, including other epoxides, based on the loss of a second methanol molecule upon CAD of the addition/methanol elimination product. The only other analytes found to undergo this elimination are some amides but they also lose O = B-R (R = group bound to carbonyl), which allows their identification. On the other hand, other epoxides can be differentiated from vinyl and phenyl epoxides and from other monofunctional analytes based on the loss of (CH₃O)₂BOH or formation of protonated (CH₃O)₂BOH upon CAD of the addition/methanol elimination product. For propylene oxide and 2,3-dimethyloxirane, the (CH₃O)₂BOH fragment is more basic than the hydrocarbon fragment, and the diagnostic ion (CH₃O)₂BOH₂⁺ is formed. These reactions involve opening of the epoxide ring. The only other analytes found to undergo (CH₃O)₂BOH elimination are carboxylic acids, but they can be differentiated from the rest based on several published ion/molecule reaction methods. Similar results were obtained in the Fourier-transform ion cyclotron resonance and linear quadrupole ion trap mass spectrometer.     

用于检测多硫化氢和亚硝酰氢的小分子荧光探针

多硫化氢（H₂S_n）和亚硝酰氢（HNO）在一系列生理病理过程中起着重要的作用,包括调节细胞内氧化还原信号传递过程、增强心肌的收缩能力、抑制血小板聚集等。H₂S_n可以通过硫化氢（H₂S）与活性氧物种反应得到。一氧化氮（NO）和HNO可以在超氧化物歧化酶（SOD）作用下相互转化,H₂S和NO反应可以生成H₂S_n和HNO,调控酶的活性以及蛋白与蛋白之间的相互作用,从而影响蛋白质的生理功能。因此,实时检测生物体内H₂S_n和HNO的浓度具有十分重要的生物医学意义。在各种生物检测技术中,荧光探针具有选择性好,灵敏度高,可以实时原位检测,对样品损伤小等优点,受到了广泛关注。本文将按照探针响应基团的反应类型,将近几年用于定性定量检测H₂S_n和HNO荧光探针进行分类和总结,重点概述探针的设计理念、响应机制和生物应用,并对探针的应用前景进行了展望。同时,本文也关注了硫化氢和其他硫烷硫类物种荧光检测的近期进展。     

A Versatile New Paradigm for the Design of Optical Nanosensors Based on Enzyme-Mediated Detachment of Labeled Reporters: The Example of Urea Detection

Here, a new bio-inspired nanoarchitectonics approach for the design of optical probes is presented. It is based on nanodevices that combine 1) an enzymatic receptor subunit, 2) a signaling subunit (consisting of a labeled reporter attached to a silica surface), and 3) a mechanism of communication between the two sites based on the production of chemical messengers by the enzymatic subunit, which induces the detachment of the reporter molecules from the silica surface. As a proof of concept, a urea nanosensor based on the release of Alexa-Fluor-647-labeled oligonucleotide from enzyme-functionalized Janus gold–mesoporous-silica nanoparticles (Au–MSNPs) was developed. The Janus particles were functionalized on the silica face with amino groups to which the labeled oligonucleotides were attached by electrostatic interactions, whereas the gold face was used for grafting urease enzymes. The nanodevice was able to release the fluorescent oligonucleotide through the enzyme-mediated hydrolysis of urea to ammonia and the subsequent deprotonation of amino groups on the silica face. This simple nanodevice was applied for the fluorometric detection of urea in real human blood samples and for the identification of adulterated milk. Given the large variety of enzymes and reporter species that could be combined, this is a general new paradigm that could be applied to the design of a number of optical probes for the detection of target analytes.     

Structure and Dynamics of Ternary Complexes of Cucurbit[8]uril with Spin-Labeled Indicators and Biologically Active Analytes

Ternary host–guest complexes have been first obtained from cucurbituril CB[8] as a host molecule and two guest molecules: nitroxyl probes of different structures and biologically important amino acids (AA) and aromatic compounds. To characterize the binding of the guests, parameters of the polarity of the environment and the rotational mobility of the spin probes have been used. These parameters have been shown to depend on the nature of the analytes. For the ternary complexes, in addition to the usual triplet ESR spectra from nitroxyl probes (S3), supramolecular ensembles consisting of three equivalent ternary complexes (“triads”) have been found, whose ESR spectra have a seven-component hyperfine structure (S7) due to delocalization of the unpaired electron over three nitrogen nuclei. The relative intensity of the S7 spectra increases with increasing NaCl concentration in the solution, and also depends significantly on the nature of the analyte and the spin probe. Quantum chemical calculations have shown that (1) to determine the stability of the complexes, it is necessary to allow for the van der Waals interaction, and (2) the complexes involving the zwitterionic form of AA are much more stable than those with the neutral form of AA.     

Dual-emissive Iridium(III) Complexes as Phosphorescent Probes with Orthogonal Responses to Analyte Binding and Oxygen Quenching

Phosphorescent probes often show sensitive response toward analytes at a specific wavelength. However, oxygen quenching usually occurs at the same wavelength and thus hinders the accurate detection of analytes. In this study, we have developed dual-emissive iridium(III) complexes that exhibit phosphorescence responses to copper(II) ions at a wavelength distinct from that where oxygen quenching occurs. The complexes displayed colorimetric phosphorescence response in aqueous solutions under different copper(II) and oxygen conditions. In cellular imaging, variation in oxygen concentration over a large range from 5 % to 80 % can modulate the intensity and lifetime of green phosphorescence without affecting the response of red phosphorescence toward intracellular copper(II) ions.