Effect of phospholipid insertion on arrayed polydiacetylene biosensors

Micro-arrayed polydiacetylene (PDA) vesicles mixed with phospholipids on glass slides were prepared for label-free detection of Escherichia coli. When E. coli bound to its antibodies chemically attached to polydiacetylene, the fluorescence of the vesicles was dramatically increased. The insertion of dimyristoyl phosphatidylcholine (DMPC) in the vesicles drastically reduced the response time for the fluorescence changes. Vesicles with 20-30% DMPC provided optimal results for bacterial detection. Fourier transform infrared (FTIR) spectra analysis suggested that DMPC insertion decreased the strength of hydrogen bonding among the amide and carboxylic acid groups of the polydiacetylene vesicles. Reduced bonding strength resulted in less rigid structure of the polydiacetylene polymer, allowing more rapid detection upon molecular recognition.     

Selective detection of catecholamines by synthetic receptors embedded in chromatic polydiacetylene vesicles

A new detection scheme for catecholamines was constructed through embedding synthetic receptors within vesicles comprising phospholipids and polydiacetylene. Fluorescence emission of the polydiacetylene was induced through specific interactions between the soluble ligands and the vesicle-incorporated hosts. The system demonstrated remarkable selectivity among structurally similar ligands and achieved much lower detection thresholds compared to that of other reported catecholamine sensors. The chromatic assembly provides a generic route for high sensitivity detection of ligand-receptor interactions.     

聚联乙炔囊泡负载的Bola型两亲分子识别三聚氰胺过程中Chaotrope促进型显色机制及热力学

用聚联乙炔囊泡为载体,将bola型两亲分子1,12-二乳清酸十二胺盐(DDO)对三聚氰胺的分子识别作用用肉眼可见的颜色变化显示出来.通过比较不同碳链长度的聚联乙炔囊泡对分子识别过程的反映,发现二十三烷基-2.4-二炔酸(TCDA)囊泡的显色灵敏度较高.研究表明,TCDA肉眼可见的颜色变化来自于DDO与三聚氰胺多重氢键的形成以及溶液环境中水结构的变化.为了更好地理解显色机理,用差示扫描量热(DSC)仪详细研究了分子识别过程中聚联乙炔囊泡的相变行为及热力学参数.结果表明:TCDA囊泡和带有识别分子的DDO/TCDA囊泡在三聚氰胺存在下,相变温度Tm均向高温方向移动,并且,随三聚氰胺浓度的增加,Tm值逐渐增大直至囊泡瓦解;但是Tm值的变化没有与囊泡变色必然关联,仅仅DDO/TCDA囊泡具有变色现象,而且,只有当三聚氰胺的浓度超过分子识别氢键形成所需理论量时,肉眼才能可见明显的由蓝到红的颜色变化.为了理解溶液中过量的三聚氰胺对囊泡变色的作用,选用蔗糖和尿素作为典型的水结构促进剂和水结构破坏剂(chaotrope),详细研究了它们对聚联乙炔囊泡反映分子识别过程中相变温度的影响及显色规律.结果表明,过量的三聚氰胺在溶液中起到类似尿素水结构破坏剂的作用.这种作用和分子识别过程中多重氢键的形成对聚联乙炔囊泡的变色缺一不可.本研究首次揭示了由水结构破坏剂参与的聚联乙炔囊泡变色机理,有助于理解共轭聚合物热相变过程中的Hofmeister效应.     

Colorimetric detection of oligonucleotides using a polydiacetylene vesicle sensor

A new system for the colorimetric detection of oligonucleotides was developed using polydiacetylene vesicles, which play the dual role of an indicator of color transition and an amplification tag. The results are of significance in understanding the mechanism of color transition of biological recognition in polydiacetylene systems and in designing new biosensors.     

Chromatic immunoassay based on polydiacetylene vesicles

A new approach of chromatic immunoassay based on polydiacetylene vesicles is described. Antibodies were covalently coupled with mixed vesicles of 10,12-tricosadiynoic acid (TCDA) and dimyristoylphosphatidycholine (DMPC). The vesicle-antibody conjugates were irradiated with UV light to yield a blue-colored polydiacetylene. After antigen injection, specific immunoreactions took place at the vesicle surface alter polydiacetylene conformation and lead to a color change from blue to red. The chromatic immunoassay described here is simple, rapid, sensitive; the color change was readily discernible by naked eye when the concentration of antigen is 1 ng/mL. Incorporation of DMPC in the mixed vesicles increases the sensitivity of the chromatic immunoassay.     

Assembly of polydiacetylene vesicles on solid substrates

A method to create assembly of polydiacetylene vesicles on solid substrates in a novel fashion is described. The formation of the assembly is based on electrostatic layer-by-layer deposition using negatively charged 10,12-pentacosadiynoic acid (PCDA) vesicles and polyelectrolyte polyethylenimine, or positively charged PCDA-2(')-aminoethylamide (PCDANH2) vesicles. This is an efficient method for preparing the chromatic sensor films of polydiacetylene vesicles after ultraviolet light irradiation. The strategy would be useful in the development of polydiacetylene-based chemosensors and biosensors.     

Color fingerprinting of proteins by calixarenes embedded in lipid/polydiacetylene vesicles

"Naked eye" color detection of proteins was achieved by embedding calixarene receptors within vesicles comprising phospholipids and the chromatic polymer polydiacetylene. Dramatic visible absorbance changes were induced through electrostatic interactions between the protein surface and the vesicle-incorporated hosts. The colorimetric responses could be induced by micromolar protein concentrations, and furthermore, specific protein fingerprints could be obtained by incorporating different receptors within the vesicles. Fluorescence and circular dichroism experiments confirmed the relationship between the colorimetric phenomena and protein docking on the surface of the chromatic vesicles. The colorimetric assay constitutes a generic platform for high-sensitivity detection of soluble proteins and for evaluation of protein surface charge distribution.     

Microbead-assisted PDA sensor for the detection of genetically modified organisms

A simple and sensitive approach for the detection of marker protein, phosphinothricin acetyltransferase, from genetically modified crops was developed based on the colorimetric transition of polydiacetylene (PDA) vesicles in combination with silica microbeads. PDAs have attracted a great deal of interests as a transducing material due to their special features that allow colorimetric response to sensory signals, as well as their inherent simplicity. However, most PDA-based biosensors require additional analytical equipment such as a fluorescence microscope or UV–Vis spectrometer. In this study, we report a new approach to increase the degree of color transition by coupling antibody-conjugated PDA vesicles with silica microbeads in an effort to monitor the results with the unaided eye or simple RGB analysis. By immobilizing PDA vesicles on silica microbeads, we were able to overcome the disadvantages of colloidal PDA-based sensors and increase the degree of colorimetric changes in response to target molecules to a concentration as low as 20 nM. The additional stresses were given to PDA vesicles by antigen–antibody bridging of PDA vesicles coupled with microbeads, resulting in enhanced blue–red color transition. All the results showed that PDA vesicles in conjunction with silica microbeads will be a promising transducing material for the detection of target proteins in diagnostic and biosensing applications.     

Physicochemical characterization of natural-like branched-chain glycosides toward formation of hexosomes and vesicles

Synthetic branched-chain glycolipids have become of great interest in biomimicking research, since they provide a suitable alternative for natural glycolipids, which are difficult to extract from natural resources. Therefore, branched-chain glycolipids obtained by direct syntheses are of utmost interest. In this work, two new branched-chain glycolipids are presented, namely, 2-hexyldecyl β(α)-D-glucoside (2-HDG) and 2-hexyldecyl β(α)-D-maltoside (2-HDM) based on glucose and maltose, respectively. The self-assembly properties of these glycolipids have been studied, observing the phase behavior under thermotropic and lyotropic conditions. Due to their amphiphilic characteristics, 2-HDG and 2-HDM possess rich phase behavior in dry form and in aqueous dispersions. In the thermotropic study, 2-HDG formed a columnar hexagonal liquid crystalline phase, whereas in a binary aqueous system, 2-HDG formed an inverted hexagonal liquid crystalline phase in equilibrium with excess aqueous solution. Furthermore, aqueous dispersions of the hexagonal liquid crystal could be obtained, dispersions known as hexosomes. On the other hand, 2-HDM formed a lamellar liquid crystalline phase (smectic A) in thermotropic conditions, whereas multilamellar vesicles have been observed in equilibrium with aqueous media. Surprisingly, 2-HDM mixed with sodium dodecyl sulfate or aerosol OT induced the formation of more stable unilamellar vesicles. Thus, the branched-chain glycolipids 2-HDG and 2-HDM not only provided alternative nonionic surfactants with rich phase behavior and versatile nanostructures, but also could be used as new drug carrier systems in the future.     

Vesicular polydiacetylene sensor for colorimetric signaling of bacterial pore-forming toxin

A vesicle-based polydiacetylene biosensor for colorimetric detection of bacterial pore-forming toxin streptolysin O (SLO) is reported. The sensor was constructed with three lipid constituents: glycine-terminated diacetylene lipid Gly-PCDA, cell membrane-mimicking component PC-DIYNE, and cholesterol (CHO), which serves as the bait molecule. UV irradiation led to photopolymerization of the diacetylene lipids that gave the material a blue appearance. Incubation of the vesicles with SLO from Streptococcus pyrogenes turned the vesicle solution red, and the color change was found to be correlated to SLO concentration. The optimal sensing performance was found with vesicles consisting of 71% Gly-PCDA, 25% CHO, and 4% PC-DIYNE, and a correlation relationship was obtained for 20 HU to 500 HU/mL, or 100 pM to 6.3 nM of SLO toxin. Transmission electron microscopy and dynamic light scattering was used for further characterization of the vesicular assemblies. Transmembrane pores (holes) with diameter around 30 nm were observed on the vesicle membranes, in particular on the peripheral of the membrane structures, suggesting pore formation by SLO toxin provides the driving force for the color change of the functional vesicles.     

One-step immobilization of alkanethiol/glycolipid vesicles onto gold electrode: amperometric detection of Concanavalin A

Functionalized vesicles composed of glycolipid and alkanethiol lipids have been immobilized onto gold surface through one-step self-assembly to construct an electrochemical biosensor for Concanavalin A (Con A) detection. Incorporation of alkanethiol lipid molecules into the vesicles allows for firm attachment of the vesicles onto a gold surface to form a sensing interface. At the same time, the introduction of alkanethiol lipid avoids cumbersome organic syntheses of sulfur-containing compound, making the biosensor greater applied prospect. Through the recognition of Con A by glycolipid which was immobilized on the surface of electrode, a decrease of electrochemical signal was observed. This decrease was restored when the electrode was immersed in a stronger binding solution such as glucose. The repeated usability of the novel sensor is excellent.     

A nanoscale vesicular polydiacetylene sensor for organic amines by fluorescence recovery

We report a nanoscale lipid membrane-based sensor of conjugated polydiacetylene (PDA) vesicles for fluorescence detection of organic amines. The vesicle sensor was constructed by incorporation of a BODIPY fluorescent dye into the PDA vesicles. The fluorescent properties of the resulting vesicles can be manipulated by adjusting lipid components, and are controlled by environmental and solution conditions. The fluorescence of the BODIPY dye was significantly quenched in the polymerization of diacetylene lipid vesicles by a UV irradiation process. However, it was sufficiently recovered by external stimuli such as a hike of solution pH. The fluorescence recovery process was reversible, and a decrease in solution pH resulted in repeated quenching. The reported system transforms an external stimulus into a large fluorescence intensity change, demonstrating great potential in developing new signal reporting method for biosensor design. The quench-recovery phenomenon of the BODIPY-PDA is believed to be related to the energy transfer between the dye and the PDA conjugate backbone. The vesicle sensor was applied for detecting an organic amine, triethylamine (TEA) and a large linear relationship was obtained between the increase in fluorescence intensity and the concentrations of TEA. The detection limit of TEA by vesicle sensors using fluorescence recovery was found to be 10 μM.     

Supramolecular chirality formation of bisazobenzene-substituted polydiacetylene LB films

In order to investigate the exact effect of stereoregular packing of head group in the side chain on the helical structure formation of polydiacetylene backbone, the larger size of bisazobenzene-substituted diacetylene monomer, 4-(4-nitrophenylazo) azobenzene-10, 12-pentacosadiynoate (BNADA) was synthesized successfully. Owing to overcrowded packing of bisazobenzene chromophores, the BNADA Langmuir-Blodgett (LB) films showed macroscopic supramolecular chirality, although BNADA molecules were achiral. Under circularly polarized UV light (CPUL) irradiation, supramolecular helix of bisazobenzene chromophores always maintained, due to the large size and lower photo-isomerization rate of bisazeobenzene chromophores. While for polydiacetylene backbone, the helical direction of the polymer chain should be decided by the competition of the effect of stereoregular packing of bisazobenzene chromophores and the interaction between the CPUL and the diacetylene dimer.     

联乙炔囊泡——一种基于分子组装的生物分子识别器件

有序排列的联乙炔分子,在紫外光照射下发生聚合。聚联乙炔（PDA）线性骨架的离域π电子,在可见光区产生π-π* 跃迁,显示特有的蓝色。聚联乙炔囊泡表面的分子探针（受体）在遇到可识别的生物大分子（如DNA,抗体-抗原和细菌等）的配体时,可使其颜色从蓝色转变为红色,有时能产生荧光。这种囊泡将检测与显示集为一体,是一种典型的生物分子器件,近年来已被用于很多物质的测定,其中包括病毒、细菌、亲脂性酶、抗菌肽类、哺乳动物肽类、离子、抗体、蛋白质和寡核苷酸等。此外,聚合囊泡还可以与金结合形成空心金球,可以作为一种三维纳米金的载体,对DNA的固定、识别和分离具有极为诱人的应用前景。通过对各种影响因素的研究和检测条件的改进,聚联乙炔生物传感器的灵敏度和选择性还能进一步提高,具有广阔的应用前景。     

Polydiacetylene/Anti‐HBs Complexes for Visible and Fluorescent Detection of Hepatitis B Surface Antigen on a Nitrocellulose Membrane

The immunochromatographic assay (ICA) using a nitrocellulose (NC) membrane offers several advantages. This technique is a rapid and straightforward method in contrast to other immunoassays. Polydiacetylene (PDA) vesicles have unique optical properties, displaying red color and red fluorescence at the same time. In this system, red‐phase PDA vesicles are used as a fluorescent dye as well as a surface for immobilized hepatitis B surface antibody (HBsAb). PDA has a remarkable stability compared with other fluorescent dyes. In this study, the most suitable PDA/HBsAb complexes are introduced for detecting hepatitis B surface antigen (HBsAg). Then, the PDA/HBsAb complexes affixed antibody is attached to NC membrane, which has two lines to confirm detection of HBsAg. The main advantage of this system is that the detection of HBsAg can be observed in both visible and fluorescent images due to the optical properties of polydiacetylene. Detection of HBsAg is observed up to 0.1 ng mL⁻¹ by fluorescent analysis and confirmed by red line on the NC membrane up to 1 ng mL⁻¹ (HBsAg) using the naked eye. Consequently, these results show that PDA/HBsAb complexes were successfully applied to ICA for the diagnosis of hepatitis B.     

One-step synthesis of amino-dextran-protected gold and silver nanoparticles and its application in biosensors

A sensitive method for the detection of the lectin protein concanavalin A (Con A) was developed using amino-dextran (AD)-protected gold (AD-Au) and silver nanoparticles (AD-Ag) as sensitive optical probes. The AD-Au and AD-Ag nanoparticles were synthesized by directly applying amino-dextran as a reductive and protective reagent. The size of the nanoparticles could be altered by changing the molar ratio of AD to the metal salt. The amino-dextran bound to Con A by forming a 4:1 Au-Con A complex at neutral pH, and the nanoparticles were induced to aggregate by Con A. The absorption intensity of the nanoparticles decreased linearly with as the Con A concentration was increased from 3.85×10^–8 to 6.15×10^–7 M. The Au-Con A complex was dissociated by the disaccharide isomaltose, which has a higher affinities for Con A than Au; this competitive strategy could also be used to detect similar types of saccharides.     

Lipopolysaccharide identification with functionalized polydiacetylene liposome sensors

The outer membrane of Gram negative bacteria contains lipopolysaccharides, which are glycolipids with carbohydrate sequences that are unique for each bacterial species and serotype. In this communication, we report a method for identifying LPS from different bacteria using an electronic tongue approach. Two functionalized polydiacetylene liposomes were used as colorimetric sensors for detecting various types of LPS. These liposomes were assayed under four different experimental conditions to generate a data set of eight colorimetric responses. This data set constitutes a unique fingerprint for each analyte and permits identification of each LPS type.     

Effect of amphiphilic molecules upon chromatic transitions of polydiacetylene vesicles in aqueous solutions

Effect of amphiphilic molecules upon the chromatic transitions of polymerized 10,12-pentacosadiynoic acid (PCDA) vesicles in aqueous solutions was reported. The colorimetric response of polymerized PCDA vesicles for 1-pentanol is higher than that for ethanol due to more hydrophobic property of 1-pentanol. The colorimetric response of polymerized PCDA vesicles for sodium dodecyl sulfate (SDS) and Triton X-100 is lower than that for cetyltrimethylammonium bromide (CTAB). The strong ability of CTAB to induce chromatic transition of the vesicles is related to the positively charged headgroups of CTAB, which favors approach of CTAB to the negatively charged carboxylate groups at the vesicle surface. The insertion of alkyl chain of CTAB into the hydrophobic domain perturbs the conformation of the conjugated polymer backbone and induces color change of polydiacetylene vesicles. For a series of alkylamine hydrochloric salts, the longer the alkyl chain, the stronger the ability of alkylamine to induce chromatic transition of polydiacetylene vesicles.     

Membrane curvature effects on glycolipid transfer protein activity

The glycolipid transfer protein (GLTP) is monomeric in aqueous solutions, and it binds weakly to membrane interfaces with or without glycolipids. GLTP is a surface-active protein and adsorbs to exert a maximal surface pressure value of 19 mN/m. The change in surface pressure following GLTP adsorption decreased linearly with initial surface pressure. The exclusion pressure for different phospholipids and sphingolipids was between 23 and 31 mN/m, being clearly highest for the negatively charged dipalmitoyl-phosphatidylserine. This can be explained by electrostatic forces when GLTP is positively charged at neutral pH (isoelectric point = 9.0) and by phosphatidylserine being negatively charged. If GLTP is injected under a palmitoyl-galactosylceramide monolayer above 30 mN/m, the presence of GLTP leads to a decrease in the surface pressure as a function of time. This suggests that GLTP is able to remove glycolipids from the monolayer without penetrating the monolayer. On the other hand, if phospholipid vesicles with or without glycolipids are also present in the subphase, no change in the surface pressure takes place. This suggests that GLTP in the presence of curved membranes is not able to transfer from or to planar membranes. We also show that transfer of fluorescently labeled galactosylceramide is faster from small highly curved palmitoyl-oleoyl-phosphatidylcholine and dipalmitoyl-phosphatidylcholine bilayer vesicles but not from palmitoyl-sphingomyelin vesicles regardless of the size.     

Dynamic light scattering as an efficient tool to study glyconanoparticle-lectin interactions

Glyconanomaterials, an emerging class of bio-functional nanomaterials, have shown promise in detecting, imaging and targeting proteins, bacteria, and cells. In this article, we report that dynamic light scattering (DLS) can be used as an efficient tool to study glyconanoparticle (GNP)--lectin interactions. Silica and Au nanoparticles (NPs) conjugated with D-mannose (Man) and D-galactose (Gal) were treated with the lectins Concanavalin A (Con A) and Ricinus communis agglutinin (RCA(120)), and the hydrodynamic volumes of the resulting aggregates were measured by DLS. The results showed that the particle size grew with increasing lectin concentration. The limit of detection (LOD) was determined to be 2.9 nM for Con A with Man-conjugated and 6.6 nM for RCA(120) with Gal-conjugated silica NPs (35 nm), respectively. The binding affinity was also determined by DLS and the results showed 3-4 orders of magnitude higher affinity of GNPs than the free ligands with lectins. The assay sensitivity and affinity were particle size dependent and decreased with increasing particle diameter. Because the method relies on the particle size growth, it is therefore general and can be applied to nanomaterials of different compositions.