Design of Turn-On Near-Infrared Fluorescent Probes for Highly Sensitive and Selective Monitoring of Biopolymers

Simple, sensitive, and selective detection of specific biopolymers is critical in a broad range of biomedical and technological areas. We present a design of turn-on near-infrared (NIR) fluorescent probes with intrinsically high signal-to-background ratio. The fluorescent signal generation mechanism is based on the aggregation/de-aggregation of phthalocyanine chromophores controlled by selective binding of small-molecule “anchor” groups to a specific binding site of a target biopolymer. As a proof-of-concept, we demonstrate a design of a sensor for EGFR tyrosine kinase—an important target in cancer research. The universality of the fluorescent signal generation mechanism, as well as the dependence of the response selectivity on the choice of the small-molecule “anchor” group, make it possible to use this approach to design reliable turn-on NIR fluorescent sensors for detecting specific protein targets present in the low-nanomolar concentration range.     

Design of Turn‐On Near‐Infrared Fluorescent Probes for Highly Sensitive and Selective Monitoring of Biopolymers

Simple, sensitive, and selective detection of specific biopolymers is critical in a broad range of biomedical and technological areas. We present a design of turn‐on near‐infrared (NIR) fluorescent probes with intrinsically high signal‐to‐background ratio. The fluorescent signal generation mechanism is based on the aggregation/de‐aggregation of phthalocyanine chromophores controlled by selective binding of small‐molecule “anchor” groups to a specific binding site of a target biopolymer. As a proof‐of‐concept, we demonstrate a design of a sensor for EGFR tyrosine kinase—an important target in cancer research. The universality of the fluorescent signal generation mechanism, as well as the dependence of the response selectivity on the choice of the small‐molecule “anchor” group, make it possible to use this approach to design reliable turn‐on NIR fluorescent sensors for detecting specific protein targets present in the low‐nanomolar concentration range.     

Harnessing Effective Molarity to Design an Electrochemical DNA‐based Platform for Clinically Relevant Antibody Detection

Easy‐to‐use platforms for rapid antibody detection are likely to improve molecular diagnostics and immunotherapy monitoring. However, current technologies require multi‐step, time‐consuming procedures that limit their applicability in these fields. Herein, we demonstrate effective molarity‐driven electrochemical DNA‐based detection of target antibodies. We show a highly selective, signal‐on DNA‐based sensor that takes advantage of antibody‐binding‐induced increase of local concentration to detect clinically relevant antibodies in blood serum. The sensing platform is modular, rapid, and versatile and allows the detection of both IgG and IgE antibodies. We also demonstrate the possible use of this strategy for the monitoring of therapeutic monoclonal antibodies in body fluids. Our approach highlights the potential of harnessing effective molarity for the design of electrochemical sensing strategies.     

Voltage-controlled metal binding on polyelectrolyte-functionalized nanopores

Most of the research in the field of nanopore-based platforms is focused on monitoring ion currents and forces as individual molecules translocate through the nanopore. Molecular gating, however, can occur when target analytes interact with receptors appended to the nanopore surface. Here we show that a solid state nanopore functionalized with polyelectrolytes can reversibly bind metal ions, resulting in a reversible, real-time signal that is concentration dependent. Functionalization of the sensor is based on electrostatic interactions, requires no covalent bond formation, and can be monitored in real time. Furthermore, we demonstrate how the applied voltage can be employed to tune the binding properties of the sensor. The sensor has wide-ranging applications and, its simplest incarnation can be used to study binding thermodynamics using purely electrical measurements with no need for labeling.     

Surface plasmon resonance sensors for real-time detection of cyclic citrullinated peptide antibodies

Surface plasmon resonance (SPR) sensors have been used for detection of various biomolecules because of their simplicity, high specificity and sensitivity, real-time detection, low cost, and no requirement of labeling. Recently, molecularly imprinted polymers that are easy to prepare, less expensive, stable, have talent for molecular recognition and also are used for creation selective binding sites for target molecule on the SPR sensors. Here, we show that preparation of cyclic citrullinated peptide antibody (anti-CCP) imprinted SPR sensor to detect CCP antibodies. For this purpose, anti-CCP/AAm pre-complex was synthesized by interacting acrylamide (AAm) monomer with anti-CCP. Then, anti-CCP imprinted (anti-CCP/PAAm) SPR sensor was obtained by reacting with anti-CCP/AAm pre-complex in the presence of the crosslinker, and initiator/activator pair. Besides this, non-imprinted (PAAm) SPR sensor was also prepared without using anti-CCP template. The SPR sensors were characterized and then adsorption-desorption studies were performed with pH 7.0 phosphate buffer (10 mM) and acetic acid (10%) with Tween 20 (1%) in pH 7.0 phosphate buffer. Selectivitiy of sensors was investigated by using immunoglobulin M (IgM) and bovine serum albumin (BSA). To determine the adsorption model of interactions between anti-CCP solutions and anti-CCP/PAAm SPR sensor, different adsorption models were performed. The calculated maximum reflection, detection limit, association and dissociation constants were 1.079 RU/mL, 0.177 RU/mL, 0.589 RU/mL and 1.697 mL/RU, respectively. Repeatability experiments of anti-CCP/PAAm SPR sensor was performed four times with adsorption-desorption-regeneration cycles without any performance losing. Results showed that anti-CCP/PAAm SPR sensor had high selectivity and sensitivity for detection of CCP antibodies.     

A selective adenosine sensor derived from a triplex DNA aptamer

The aim of this study is to develop a selective adenosine aptamer sensor using a rational approach. Unlike traditional RNA aptamers developed from SELEX, duplex DNA containing an abasic site can function as a general scaffold to rationally design aptamers for small aromatic molecules. We discovered that abasic site-containing triplex DNA can also function as an aptamer and provide better affinity than duplex DNA aptamers. A novel adenosine aptamer sensor was designed using such a triplex. The aptamer is modified with furano-dU in the binding site to sense the binding. The sensor bound adenosine has a dissociation constant of 400 nM, more than tenfold stronger than the adenosine aptamer developed from SELEX. The binding quenched furano-dU fluorescence by 40%. It was also demonstrated in this study that this sensor is selective for adenosine over uridine, cytidine, guanosine, ATP, and AMP. The detection limit of this sensor is about 50 nM. The sensor can be used to quantify adenosine concentrations between 50 nM and 2 μM.     

Selective recognition of organic pollutants in aqueous solutions with composite imprinted membranes

In this work, thin-layer composite membranes imprinted with desmetryn or ibuprofen were prepared and studied for selective recognition of the template compounds in aqueous solutions. The imprinted membranes were developed using photoinitiated copolymerization of 2-acrylamido-2-methyl-1-propane sulphonic acid and N,N'-methylene-bis-acrylamide, in the presence of desmetryn or via copolymerization of dimethylaminoethyl methacrylate, and trimethylopropane trimethacrylate, in the presence of ibuprofen, followed by deposition of the imprinted layers on the surface of porous microfiltration supports of various chemical nature. Atomic force microscopy was used to study the surface morphological characteristics of the developed membranes. Molecularly recognition properties of imprinted membranes were evaluated by measuring their capability to bind the template molecules from polycomponent aqueous solutions. It was shown that obtained membranes may be used as selective recognising elements of portative differential capacitor sensor device for express monitoring of the target molecules in water. The sensor performance is based on registration of the alteration of dielectric permeability of composite imprinted membrane at selective binding of template molecules, when the analyzed feed solution is filtered through the membrane sample.     

A bifunctional rhodamine derivative as chemosensor for recognizing Cu~(2+) and Hg~(2+) ions via different spectra

A new simple bifunctional chemosensor 1 based on rhodamine was synthesized by hydrazide and formylformic acid,which could detect Cu~(2+) and Hg~(2+) via dif ferent detecting methods in CH3 CN-HEPES buffer solution(20 mmol/L,pH 7.4)(1:9,v/v) respectively.When sensor 1 bound with Cu~(2+),it showed a colorimetric change,while a selective enhancement in fluorescence occurred upon 1 binding with Hg~(2+),resulting from the spirolatam-ring opening process.The binding modes of 1 with Cu~(2+) and Hg~(2+) were investigated based on UV,fluorescence change,ESI-Mass and Job's Plot data.Moreover,sensor 1 could selectively detect target ion in a mixed solution of Cu~(2+) and Hg~(2+),and the two metal ions do not inte rfere with each other in the process of detecting Cu~(2+) or Hg~(2+) with 1.     

Context-dependent fluorescence detection of a phosphorylated tyrosine residue by a ribonucleopeptide

Tools for selective recognition and sensing of specific phosphorylated tyrosine residues on the protein surface are essential for understanding signal transduction cascades in the cell. A stable complex of RNA and peptide, a ribonucleopeptide (RNP), provides effective approaches to tailor RNP receptors and fluorescent RNP sensors for small molecules. In vitro selection of an RNA-derived pool of RNP afforded RNP receptors specific for a phosphotyrosine residue within a defined amino-acid sequence Gly-Tyr-Ser-Arg. The RNP receptor for the specific phosphotyrosine residue was successfully converted to a fluorescent RNP sensor for sequence-specific recognition of a phosphorylated tyrosine by screening a pool of fluorescent phosphotyrosine-binding RNPs generated by a combination of the RNA subunits of phosphotyrosine-binding RNPs and various fluorophore-modified peptide subunits. The phosphotyrosine-binding RNP receptor and fluorescent RNP sensor constructed from the RNP receptor not only discriminated phosphotyrosine against tyrosine, phosphoserine, or phosphothreonine, but also showed specific recognition of amino acid residues surrounding the phosphotyrosine residue. A fluorescent RNP sensor for one of the tyrosine phosphorylation sites of p100 coactivator showed a binding affinity to the target site ~95-fold higher than the other tyrosine phosphorylation site. The fluorescent RNP sensor has an ability to function as a specific fluorescent sensor for the phosphorylated tyrosine residue within a defined amino-acid sequence in HeLa cell extracts.     

Solution-phase single quantum dot fluorescence resonance energy transfer

We present a single particle fluorescence resonance energy transfer (spFRET) study of freely diffusing self-assembled quantum dot (QD) bioconjugate sensors, composed of CdSe-ZnS core-shell QD donors surrounded by dye-labeled protein acceptors. We first show that there is direct correlation between single particle and ensemble FRET measurements in terms of derived FRET efficiencies and donor-acceptor separation distances. We also find that, in addition to increased sensitivity, spFRET provides information about FRET efficiency distributions which can be used to resolve distinct sensor subpopulations. We use this capacity to gain information about the distribution in the valence of self-assembled QD-protein conjugates and show that this distribution follows Poisson statistics. We then apply spFRET to characterize heterogeneity in single sensor interactions with the substrate/target and show that such heterogeneity varies with the target concentration. The binding constant derived from spFRET is consistent with ensemble measurements.     

Increasing the Sensitivity and Single‐Base Mismatch Selectivity of the Molecular Beacon Using Graphene Oxide as the “Nanoquencher”

Here, we report a novel, highly sensitive, selective and economical molecular beacon using graphene oxide as the “nanoquencher”. This novel molecular beacon system contains a hairpin‐structured fluorophore‐labeled oligonucleotide and a graphene oxide sheet. The strong interaction between hairpin‐structured oligonucleotide and graphene oxide keep them in close proximity, facilitating the fluorescence quenching of the fluorophore by graphene oxide. In the presence of a complementary target DNA, the binding between hairpin‐structured oligonucleotide and target DNA will disturb the interaction between hairpin‐structured oligonucleotide and graphene oxide, and release the oligonucleotide from graphene oxide, resulting in restoration of fluorophore fluorescence. In the present study, we show that this novel graphene oxide quenched molecular beacon can be used to detect target DNA with higher sensitivity and single‐base mismatch selectivity compared to the conventional molecular beacon.     

Identification of Potent,Selective Protein Kinase C Inhibitors Based on a Phorbol Skeleton

The elucidation of specific functions of protein kinase C (PKC) subtypes in physiological processes is an important challenge for the future development of new drug targets. Subtype‐selective PKC agonists and antagonists are useful biological tools for this purpose. Most of the currently used PKC modulators elicit their activities through binding to the ATP binding site of PKC, which shares many features with other kinases. PKC modulators that target the PKC regulatory domain are considered to be advantageous in terms of selectivity, because the structure of the regulatory domain is intrinsic to each PKC subtype. In this paper, we describe the identification of new potent and conventional PKC‐selective inhibitors that target the regulatory domain. The inhibitors contain a phorbol skeleton, a naturally occurring potent and selective PKC regulatory domain binder, with a perfluorinated alkyl group and a polyether hydrophilic chain on a terephthaloyl aromatic ring at the C12 position. Both of these substituents are essential for the potent inhibitory activity. Specifically, the binding affinity between PKC and the phorbol ester analogues was improved by an electron‐deficient aromatic ring at C12. This finding cannot be explained by the previously proposed binding model and suggests a new binding mode between phorbol esters and PKC.     

A general framework for inhibitor resistance in protein kinases

Protein kinases control virtually every aspect of normal and pathological cell physiology and are considered ideal targets for drug discovery. Most kinase inhibitors target the ATP binding site and interact with residue of a hinge loop connecting the small and large lobes of the kinase scaffold. Resistance to kinase inhibitors emerges during clinical treatment or as a result of in vitro selection approaches. Mutations conferring resistance to ATP site inhibitors often affect residues that line the ATP binding site and therefore contribute to selective inhibitor binding. Here, we show that mutations at two specific positions in the hinge loop, distinct from the previously characterized "gatekeeper," have general adverse effects on inhibitor sensitivity in six distantly related kinases, usually without consequences on kinase activity. Our results uncover a unifying mechanism of inhibitor resistance of protein kinases that might have widespread significance for drug target validation and clinical practice.     

5‐Formyluracil as a Multifunctional Building Block in Biosensor Designs

In organisms 5‐formyluracil (5fU), which is known as a vital natural nucleobase, is widely present. Despite the recent development of sensor designs for organic fluorescent molecules for selective targeting applications, biocompatible and easily operated probe designs that are based on natural nucleobase modifications have rarely been reported. Here, we introduce the idea of 5fU as a multifunctional building block to facilitate the design and synthetic development of biosensors. The azide group was derived from the sugar of a nucleoside, which can be further used in the selective binding of cells or organelles through click chemistry with alkynyl‐modified targeting groups. The aldehyde group of 5fU can react with different chemicals to generate environmentally sensitive nucleobases that have obvious characteristics, which precious reactants cannot achieve for selective fluorogenic switch‐on detection of a specific target. We first synthesized 5fU analogues that had aggregation‐induced emission properties, and then we used triphenylphosphonium as a mitochondria‐targeting group to selectively image mitochondria in cancer cells and mouse embryonic stem cells. Additionally, the reagents exhibit a high selectivity for reaction with 5fU, which means that the method can also be used for the detection of 5fU. Combining the two characteristics, the idea of 5fU as a multifunctional building block in biosensor designs may potentially be applicable in 5fU site‐specific microenvironment detection in future research.     

Multidentate Lewis acids: synthesis,structure and mode of action of a redox-based fluoride ion sensor

The mode of action of the bidentate bis(boronate) Lewis acid 2 as a fluoride ion sensor is shown to involve selective anion binding together with an electrochemical response.     

Harnessing Effective Molarity to Design an Electrochemical DNA-based Platform for Clinically Relevant Antibody Detection

Easy-to-use platforms for rapid antibody detection are likely to improve molecular diagnostics and immunotherapy monitoring. However, current technologies require multi-step, time-consuming procedures that limit their applicability in these fields. Herein, we demonstrate effective molarity-driven electrochemical DNA-based detection of target antibodies. We show a highly selective, signal-on DNA-based sensor that takes advantage of antibody-binding-induced increase of local concentration to detect clinically relevant antibodies in blood serum. The sensing platform is modular, rapid, and versatile and allows the detection of both IgG and IgE antibodies. We also demonstrate the possible use of this strategy for the monitoring of therapeutic monoclonal antibodies in body fluids. Our approach highlights the potential of harnessing effective molarity for the design of electrochemical sensing strategies.     

Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells

The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells.     

Specific recognition of a nucleobase-functionalized poly(3,4-ethylenedioxithiophene) (PEDOT) in aqueous media

In this Letter we report the synthesis, characterization, and electrochemical investigation of a 3,4-ethylenedioxythiophene (EDOT) derivative covalently linked to the nucleobase uracil. The successful electrochemical polymerization of this derivative has provided modified electrodes with a novel functional poly(3,4-ethylenedioxythiophene) derivative. Recognition experiments in aqueous media have shown the specific recognition of the complementary base adenine. The electrochemical detection of the selective binding of nucleobases to this PEDOT derivative in aqueous media can be of particular interest for electrochemical sensor applications in physiological media.     

The first fluorescent sensor for D-glucarate based on the cooperative action of boronic acid and guanidinium groups

A new fluorescent sensor (1) with a recognition unit consisting of a boronic acid moiety and a guanidinium unit shows selective binding of D-glucarate in aqueous solution.     

A fluorescence turn-on sensor for iodide based on a thymine-Hg(II)-thymine complex

Iodide plays a vital role in many biological processes, including neurological activity and thyroid function. Due to its physiological relevance, a method for the rapid, sensitive, and selective detection of iodide in food, pharmaceutical products, and biological samples such as urine is of great importance. Herein, we demonstrate a novel and facile strategy for constructing a fluorescence turn-on sensor for iodide based on a T-Hg(II)-T complex (T=thymine). A fluorescent anthracene-thymine dyad (An-T) was synthesized, the binding of which to a mercury(II) ion lead to the formation of a An-T-Hg(II)-T-An complex, thereby quenching the fluorescent emission of this dyad. In this respect, the dyad An-T constituted a fluorescence turn-off sensor for mercury(II) ions in aqueous media. More importantly, it was found that upon addition of iodide, the mercury(II) ion was extracted from the complex due to the even stronger binding between mercury(II) ions and iodide, leading to the release of the free dyad and restoration of the fluorescence. By virtue of this fluorescence quenching and recovery process, the An-T-Hg(II)-T-An complex constitutes a fluorescence turn-on sensor for iodide with a detection limit of 126 nM. Moreover, this sensor is highly selective for iodide over other common anions, and can be used in the determination of iodide in drinking water and biological samples such as urine. This strategy may provide a new approach for sensing some other anions.