Optical characteristics of responsive biopolymers; co-polycondensation of tri-functional amino acids and Cy-3 bis-amine with diacylchlorides

The synthesis and fluorescence behaviour of a range of amphiphilic poly(l-lysine co-bis-amine-Cy-3 iso-phthalamide) polymers that incorporate low levels of Cy-3 dye co-monomer (0.5-2.1% (w/w)) in their backbone have been investigated in aqueous solution over a range of pH values. The desired functionality of these biocompatible, hydrophobically modified polyelectrolytes was achieved by incorporating pendant hydrophilic carboxyl groups along the polymer backbone, via the l-lysine moiety, balanced by a degree of hydrophobicity introduced via the iso-phthaloyl moiety. Thus, the polymer changes from an extended conformation at high degrees of ionisation to a compact conformation stabilised by hydrophobic association at low degrees of ionisation and eventually precipitates from solution. At intermediate degrees of ionisation, such polymers exhibit amphiphilic properties. A bis-functional cyanine fluorophore derivative, co-polymerised within the polymer backbone, is demonstrated to act as a fluorescent reporter on the conformational state of the polymer. The materials have higher intrinsic fluorescence intensity per fluorophore monomer over a broad range of concentrations than bis-amine Cy-3 and their maximum fluorescence intensity is up to eight fold higher than the maximum intensity of bis-amine Cy-3, which is limited by quenching. It is also shown that the free fluorophore can be used to probe the conformation of unlabelled poly(l-lysine iso-phthalamide) through electrostatic interaction with the polymer. The technique allows rapid spectrophotometric determination of polymer conformation and offers the potential of an environmentally sensitive molecular pH probe for in vivo use.     

DNase-activatable fluorescence probes visualizing the degradation of exogenous DNA in living cells

This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the occurrence of a red fluorescence signal due to fluorescence resonance energy transfer (FRET). DNase in biological samples was detected using DFProbes and the fluorescence imaging in living cells was performed using DFprobe-modified Au nanoparticles. The results show that DFProbes have good responses to DNase, and can clearly visualize the degradation of exogenous DNA in cells in real time. The well-designed probes might be useful in tracing the dynamic changes of exogenous DNA and nanocarriers in vitro and in vivo.     

A “Quenchergenic” Chemoselective Protein Labeling Strategy

Dynamic changes in protein structure can be monitored by using a fluorescent probe and a dark quencher. This approach is contingent upon the ability to precisely introduce a fluorophore/quencher pair into two specific sites of a protein of interest. Despite recent advances, there is continued demand for new and convenient approaches to site-selectively label proteins with such optical probes. We have recently developed a chemoselectively rapid azo-coupling reaction (CRACR) for site-specific protein labeling; it relies on rapid coupling between a genetically encoded 5-hydroxytryptophan residue and various aromatic diazonium ions. Herein, it is reported that the product of this conjugation reaction, a highly chromophoric biarylazo group, is a potent fluorescence quencher. The absorption properties of this azo product can be tuned by systematically altering the structure of the aryldiazonium species. A particular “quenchergenic” aryldiazonium has been identified that, upon conjugation, efficiently quenches the fluorescence of green fluorescent protein, which is a widely used genetically encoded fluorescent probe that can be terminally attached to target proteins. This fluorophore/quencher pair was used to evaluate the protein-labeling kinetics of CRACR, as well as to monitor the proteolysis of a fusion protein.     

A Method to Generate and Analyze Modified Myristoylated Proteins

Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motifs, coupled with bio‐orthogonal chemistry, have been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP‐ribosylation factor 1 (Arf1). The capacities of these recombinant proteins to bind liposomes and load and hydrolyze GTP were measured and compared with the unmodified myristoylated Arf1. The ketone‐modified myristoylated Arf1 could be further labeled by fluorophore‐coupled hydrazine and subsequently visualized through fluorescence imaging. This methodology provides an effective model system to characterize lipid‐modified proteins with additional functions before applying them to cellular systems.     

Water‐soluble fluorescent nanoparticles without distinct aggregation of conjugated polymer chains

In a previous study, we reported water‐soluble light‐emitting nanoparticles with distinct interchain aggregation states of the constituent conjugated polymers. These interchain states usually result in strong self‐quenching, dramatically reducing the quantum efficiency of fluorescence. In the work reported in the present study, we developed new water‐soluble fluorescent nanoparticles without distinct aggregation of the conjugated polymer chains, which demonstrated distinctive morphologies and optical properties. ‘Strawberry’ morphologies of the nanoparticles were directly observed using transmission electron microscopy. The conjugated polymers were dispersed in the individual cores of the nanoparticles and the majority of the core diameters were in the range 8–12 nm. The primary optical properties of the conjugated polymers in tetrahydrofuran still remained in the nanoparticles. The results suggest that the conjugated polymer chains formed a possible unimolecular structure without distinct aggregation in the nanoparticles. Copyright © 2010 Society of Chemical Industry     

Rapid Estimation of Membrane Protein Orientation in Liposomes

The topological organization of proteins embedded in biological membranes is crucial for the tight interplay between these enzymes and their accessibility to substrates in order to fulfil enzymatic activity. The orientation of a membrane protein reconstituted in artificial membranes depends on many parameters and is hardly predictable. Here, we present a convenient approach to assess this important property independent of the enzymatic activity of the reconstituted protein. Based on cysteine-specific chemical modification of a target membrane protein with a cyanine fluorophore and a corresponding membrane-impermeable fluorescence quencher, the novel strategy allows rapid evaluation of the distribution of the two orientations after reconstitution. The assay has been tested for the respiratory complexes bo₃ oxidase and ATP synthase of Escherichia coli and the results agree well with other orientation determination approaches. Given the simple procedure, the proposed method is a powerful tool for optimization of reconstitution conditions or quantitative orientation information prior to functional measurements.     

Diaminoterephthalate turn-on fluorescence probes for thiols--tagging of recoverin and tracking of its conformational change

Diaminoterephthalates with a maleimide moiety were synthesized and used as fluorescence dyes for sensing thiols. Whereas these "NiWa Blue" dyes showed no emission, the conjugate addition of a thiol to the maleimide group turned on a fluorescence at about 400 nm when irradiating the dye at 338 nm. The neuronal-calcium sensor protein recoverin possesses a single cysteine residue at position 39, which reacts with NiWa Blue, and is therefore labeled by a fluorophore with an emission at about 440 nm. In the absence of Ca(2+), irradiation at 280 nm of a tryptophan residue in close proximity to Cys-bound NiWa Blue lead to strong FRET, which was detected by emission of the dye at 440 nm. In the presence of Ca(2+), the protein holds a conformation with distal Trp and Cys residues, thus FRET of irradiated Trp to Cys-bound NiWa Blue was significantly weakened.     

Fluorescence Intensity Modulation in Photochromic Conjugated Polymer Systems

The ability to modulate fluorescence intensity with a light signal enables a variety of applications based on fluorescence imaging. One approach to fluorescence photomodulation involves using a photochromic moiety that responds to a light signal in conjunction with a nearby fluorophore. We employ conjugated polymers based on poly(p-phenylene vinylene) (PPV) as the fluorophore in photochrome-fluorophore systems for fluorescence modulation. Advantages of using conjugated polymers for this purpose include their intrinsic energy migration processes that enable amplified fluorescence quenching as well as their processability. Here we present examples of PPV-based photomodulation systems that employ photochromic dyes from three common photochromic families: azobenzenes, spironaphthoxazines, and diarylethenes. In all cases we observe reversible fluorescence quenching due to fluorescence resonance energy transfer to the photogenerated form of the photochrome. Examples of the photomodulation of photochromic PPV systems in organic solution, polymer films, and conjugated polymer nanoparticles are presented.     

The unique optical behaviour of bio‐related materials with organic chromophores

Molecularly designed materials based on macromolecules and organic dyes offer unique opportunities in connection with the possibility of preparing optically responsive ‘smart’ materials. Indeed macromolecules are able to transmit and amplify small signals reaching sites at interacting distance through the involvement of the whole chain. The corresponding materials can then acquire stimuli‐responsive properties in relation to specific features connected to primary structure and conformation. As a first approach to benefit from the above features for preparing eco‐compatible smart materials, bio‐related polypeptides, polysaccharides and polyesters can be used as the macromolecular partner in combination with a selected dye following different interaction methodologies. Two distinct routes were used to prepare optically responsive products from the above bio‐related polymers, respectively based either on the covalent bonding to the original macromolecules of photochromic molecular species, such as azobenzene and spiropyran, or on the morphology‐modulated dispersion of highly conjugated dyes in the polymer bulk. Examples related to the two different routes have been investigated in our laboratory and are presented and discussed also with reference to selected recent cases from the literature. Copyright © 2012 Society of Chemical Industry     

Optical detection of DNA and proteins with cationic polythiophenes

In recent years, intense research has been carried out worldwide with the goal of developing simple, sensitive, and specific detection tools for biomedical applications. Along these lines, we reported in 2002 on cationic polythiophene derivatives able to provide ultrasensitive detection levels and the capability to distinguish perfect matches from oligonucleotides having as little as a single base mismatch. It was shown that the intrinsic fluorescence of the random-coil polymers quenches as a result of the planar, highly conjugated conformation adopted by the polymers when complexed with a single-strand DNA (ssDNA) capture probe but increases again after hybridization with the perfectly matched complementary strand. This change in fluorescence intensity is mainly due to a modification in the delocalization of pi electrons along the carbon chain backbone that occurs when switching between the two conformations. Thus, by monitoring, via the change in fluorescence intensity, the hybridization of the complementary ssDNA target with the "duplex", one could detect as little as 220 complementary target molecules in a 150 microL sample volume (0.36 zmol) in less than 1 hour. Building on this initial concept, we then reported that tagging the DNA probe with a suitable fluorophore dramatically increases the detection sensitivity. This novel molecular system involves the self-assembly of aggregates of duplexes in solution, prior to the introduction of the target, which allows a highly efficient resonance energy transfer (RET) between a "donor" (being the complex formed of the DNA double helix and the polymer chain wrapped around it) and a large number of neighboring "acceptors" (the fluorophores attached to the DNA probes). The massive intrinsic signal amplification (fluorescence chain reaction or FCR) provided by this novel integrated molecular system allows the specific detection of as little as five dsDNA copies in a 3 mL sample volume in only 5 minutes, without the need for prior amplification of the target. Clearly, direct and reliable detection of DNA hybridization without prior PCR amplification or chemical tagging of the genetic target is now possible with this methodology. We have also shown that proteins can be detected following a similar strategy. Impressive results have also been reported by direct and specific staining of targeted proteins. All these features have recently allowed the development of responsive polymeric supports for the detection of DNA and proteins. All these assays that do not require any chemical manipulation of the biological targets or sophisticated experimental procedures should soon lead to major advances in genomics and proteomics.     

Understanding of mechanistic perspective in sensing of energetic nitro compounds through spectroscopic and electrochemical studies

Understanding the probable mechanism of detection plays a significant role in selecting a particular fluorophore for the detection of the complicated and low volatile analyte namely, nitroaromatic and non-aromatic high energy materials (HEMs) in the development of detection devices when compared with the existing techniques. The fluorescence quenching employing conducting polymers-based response of HEMs have attained great significance in recent times. Amongst all conducting polymers, functionalized polyaniline can act as a fluorophore in quenching studies. This report also reveals the importance of studying electrochemical methods associated with the changes observed in the oxidation potential and its resemblance with fluorescence quenching study in establishing the probable mechanism associated with the sensing study of HEMs (viz., RDX, PETN, CL-20 and RDX: CL-20 Cocrystal). FTIR and resonance Raman characterization helped us to understand the interaction between nitro groups present in HEMs with benzenoid unit or polaronic or bipolaronic nitrogen of camphor sulfonic acid doped polyaniline (CSA[PANi]). Employing Stern Volmer plot, the efficiency of quenching and quenching mechanisms of complex structure formed between the fluorophore and HEMs is understood.     

Cleavable substrate containing molecular beacons for the quantification of DNA-photolyase activity

In order to gain deeper insight into the function and interplay of proteins in cells it is essential to develop methods that allow the profiling of protein function in real time, in solution, in cells, and in cell organelles. Here we report the development of a U-type oligonucleotide (molecular beacon) that contains a fluorophore and a quencher at the tips, and in addition a substrate analogue in the loop structure. This substrate analogue induces a hairpin cleavage in response to enzyme action, which is translated into a fluorescence signal. The molecular beacon developed here was used to characterize DNA-photolyase activity. These enzymes represent a challenge for analytical methods because of their low abundance in cells. The molecular beacon made it possible to measure the activity of purified class I and class II photolyases. Photolyase activity was even detectable in crude cell extracts.     

Conjugated polymers with tethered electron‐accepting moieties as ambipolar materials for photovoltaics

Conjugated polymers are of increasing interest as semiconductors for soft (opto)electronic devices, including photovoltaic elements. A promising conversion of solar energy into electrical energy is possible with blends of soluble electron donor‐type conjugated polymers and fullerenes as electron‐acceptor, transporting component. This approach, called bulk‐heterojunction, suggested the preparation of intrinsic ambipolar materials to control simultaneously the electronic and morphological properties. On these bases, the covalent grafting of acceptor moieties onto conjugated backbones seemed attractive for the preparation of intrinsically ambipolar polymeric materials (‘double‐cable’ polymers) as an alternative to donor–acceptor composites. The design, characterisation and application of this novel class of polymers are reviewed taking into account the current understanding of organic photovoltaics. Copyright © 2007 Society of Chemical Industry     

Structural materials underpinning functional materials: teaching old dogs new tricks

‘Structural’ polymers are finding new applications underpinning new technology developments based on functional polymers. This paper discusses the use of polyester films as base substrates for flexible electronic applications based on conjugated polymers and low‐temperature hydrogenated amorphous silica processing, and discusses the challenges involved in successfully developing substrates ‘fit for purpose’. Copyright © 2008 Society of Chemical Industry     

Ultrasensitive Molecular Beacon Designed with Totally Serinol Nucleic Acid (SNA) for Monitoring mRNA in Cells

An artificial nucleic acid based on acyclic serinol building blocks and termed “serinol nucleic acid” (SNA) was used to construct a fluorescent probe for RNA visualization in cells. The molecular beacon (MB) composed of only SNA with a fluorophore at one terminus and a quencher at the other was resistant to enzymatic digestion, due to its unnatural acyclic scaffold. The SNA‐MB could detect its complementary RNA with extremely high sensitivity; the signal‐to‐background (S/B) ratio was as high as 930 when perylene and anthraquinone were used as the fluorophore and quencher pair. A high S/B ratio was also achieved with SNA‐MB tethering the conventional Cy3 fluorophore, and this probe enabled selective visualization of target mRNA in fixed cells. Thus, SNA‐MB has potential for use as a biological tool capable of visualizing RNA in living cells.     

Fluorescence quenching of poly(methylphenylsilane) in solution

The solution, electron-transfer fluorescence quenching of a typical aromatic polysilane[poly(methylphenylsilane)] by a series of electronic deficient aromatic monomers is described. The rate of fluorescence quenching is a function of the reduction potential of the quencher, and only very fast processes can be observed, due to the short polymer fluorescence lifetime. The measurement of quenching rate constants, which are considerably larger than diffusion control, suggests that extensive energy migration occurs in the polymer. Although the fluorescence quenching at low quencher concentrations follows Stern-Volmer kinetics, at high concentrations, contributions from static quenching are apparent. Strong fluorescence quenching can either accelerate or inhibit photodegradation, depending on the structure of the quencher.     

Effect of the polysilane structure on its solution fluorescence quenching

We studied the solution fluorescence quenching of poly(methylphenethylsilane) (1^#), poly(dimethylsilane‐co‐methylphenethylsilane) (2^#), poly(n‐hexylmethylsilane) (3^#), and poly(dimethylsilane‐co‐n‐hexylmethylsilane) (4^#) by such quenchers as CCl₄, CHCl₃, Cl₂CHCHCl₂, and methyl benzoate. We treated the fluorescence quenching data using the equations F₀/F = 1 +K_SV[Q],F₀/F exp(−NV[Q]) = 1 +K_SV[Q], and ln(F₀/F) =NV[Q], where F and F₀ are the fluorescence intensity with and without the addition of a quencher, respectively; K_SV, the Stern–Volmer constant; [Q], the quencher concentration; N, Avogadro's constant; and V, the volume of the active sphere. For the systems with both static quenching and dynamic quenching, we calculated their contributions and the critical quencher concentration [Q]_C and determined the nature of the fluorescence quenching in different quencher concentration ranges. We observed that, under the condition of the same quencher, the fluorescence quenching of the polysilane homopolymer is smaller than that of its corresponding polysilane copolymer, that is, 1^# < 2^# and 3^# < 4^#, and that for the fluorescence quenching of the same polysilane by different chlorohydrocarbons the fluorescence quenching ability of CCl₄ is larger than that of CHCl₃ and Cl₂CHCHCl₂. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 133–139, 2000     

Synthetic DNA aptamers to detect protein molecular variants in a high-throughput fluorescence quenching assay

Real-time protein detection in homogeneous solutions is necessary in many biotechnology and biomedical studies. The recent development of molecular aptamers, combined with fluorescence techniques, may provide an easy and efficient approach to protein elucidation. This report describes the development of a fluorescence-based assay with synthetic DNA aptamers that can detect and distinguish molecular variants of proteins in biological samples in a high-throughput process. We used an aptamer with high affinity for the B chain of platelet-derived growth factor (PDGF), labeled it with a fluorophore and a quencher at the two termini, and measured fluorescence quenching by PDGF. The specific quenching can be used to detect PDGF at picomolar concentrations even in the presence of serum and other cell-derived proteins in cell culture media. This is the first successful application of a synthetic aptamer for the detection of tumor-related proteins directly from the tumor cells. We also show that three highly related molecular variants of PDGF (AA, AB, and BB dimers) can be distinguished from one another in this single-step assay, which can be readily adapted to a microtiter plate assay for high-throughput analysis. The use of fluorescence quenching as a measure of binding between the DNA probe and the target protein eliminates potential false signals that may arise in traditional fluorescence enhancement assays as a result of degradation of the DNA aptamer by contaminating nucleases in biological specimens. This assay is applicable to proteins that are not naturally DNA binding. The excellent specificity, ultrahigh sensitivity, and simplicity of this one-step assay addresses a growing need for high-throughput methods that detect changes in the expression of gene products and their variants in cell cultures and biological specimens.     

稳态荧光猝灭法确定胶束聚合合成条件

以芘(Py)为荧光探针,十六烷基氯化吡啶(CPC)为猝灭剂,用稳态荧光猝灭法研究十二烷基硫酸钠(SDS)浓度和溶液极性对其胶束聚集数的影响。结果表明,当SDS浓度在5～42倍cmc时,胶束聚集数Nagg随SDS的浓度增大而增大,并且趋势逐渐变缓。当SDS浓度为11.3倍cmc时,胶束聚集数随NaCl浓度增大而明显增大,I1/I3值逐渐减小。以上两种条件下聚集数的增大引起疏水微嵌段长度较大的变化,而微嵌段长度又是胶束聚合中最重要的参数之一。因此,胶束聚合合成微嵌段缔合聚合物中,SDS浓度和溶液极性对其胶束聚集数的影响不能忽略。     

Investigation of molecular beacon aptamer-based bioassay for platelet-derived growth factor detection

This report describes studies on the use of a molecular-beacon aptamer (MBA) as a synthetic high-affinity DNA probe that exhibits fluorescence resonance energy transfer (FRET) in response to a specific protein biomarker, platelet-derived growth factor (PDGF). As a step toward the application of the MBA in a fluorescence-based assay for biological specimens, we examined the influence of certain physical and chemical parameters of incubation that would affect DNA conformation and DNA-backbone modification, and thus improve nuclease resistance. This bioassay is compatible with pH, temperature, and monovalent cation levels typically encountered in biological samples, and phosphorothioate backbone-modified MBA is able to exhibit specific FRET. With minimal sample processing and without assay optimization, the MBA is able to detect as little as 10 ng PDGF per mug of serum proteins from cell-culture media. We also show that different sets of known fluorophore-quencher pairs can be successfully used in the MBA for sensitive detection of the PDGF target. It should, therefore, be possible to develop multiplex bioassays that monitor either quenching or enhancement for the simultaneous detection of several biomarkers by using MBAs created from high-affinity DNA ligands for the desired protein targets. Interestingly, we observed that, with a DNA ligand with multiple binding sites for a standard multimeric protein target, the FRET bioassay could be accomplished by using a mixture of two individually labeled DNAs-one carrying the fluorophore and the other with the matching quencher. This observation has significant implications in the future design of more selective DNA-based FRET bioassays that use more than one ligand for the same protein target.