Fluorescent nanoscale detection of biotin-streptavidin interaction using near-field scanning optical microscopy

We describe a nanoscale strategy for detecting biotin-streptavidin binding using near-field scanning optical microscopy (NSOM) that exploits the fluorescence properties of single polydiacetylene (PDA) liposomes. NSOM is more useful to observe nanomaterials having optical properties with the help of topological information. We synthesized amine-terminated 10,12-pentacosadiynoic acid (PCDA) monomer (PCDA-NH(2)) and used this derivatized monomer to prepare PCDA liposomes. PCDA-NH(2) liposomes were immobilized on an aldehyde-functionalized glass surface followed by photopolymerization by using a 254?nm light source. To measure the biotin-streptavidin binding, we conjugated photoactivatable biotin to immobilized PCDA-NH(2) liposomes by UV irradiation (365?nm) and subsequently allowed them to interact with streptavidin. We analyzed the fluorescence using a fluorescence scanner and observed single liposomes using NSOM. The average height and NSOM signal observed in a single liposome after binding were ～31.3 to 8.5 ± 0.5?nm and 0.37 to 0.16 ± 0.6?kHz, respectively. This approach, which has the advantage of not requiring a fluorescent label, could prove highly beneficial for single molecule detection technology.     

DNA-oligonucleotide encapsulating liposomes as a secondary signal amplification means

A novel liposome-based signal amplification system was developed by encapsulating DNA oligonucleotides within antibody-tagged liposomes and subsequently detecting the oligonucleotide with dye-encapsulating liposomes for double signal enhancement. In this sandwich immunoassay, the model analyte, protective antigen protein from B. anthracis, was captured by one set of antibodies immobilized in microtiter plate wells and detected using a second antibody conjugated to oligonucleotide-encapsulating liposomes. Bound liposomes were lysed releasing the encapsulated fluorescein-tagged DNA 25-mer probe, which was then permitted to hybridize with its complementary sequence immobilized in a second plate. Finally, the amount of oligonucleotide was detected through the addition of anti-fluorescein antibody tagged dye-encapsulating liposomes. These secondary liposomes allowed for a approximately 400x lower LOD than detection of the fluorescein-labeled probe alone. Several aspects were investigated, including the encapsulation of various oligonucleotide concentrations within liposomes; oligonucleotide hybridization times and buffers; degree of anti-fluorescein antibody coverage on the liposomes; and immobilized anti-protective antigen antibody concentration. We found that the encapsulation efficiency increased with the starting oligonucleotide concentration. As many as 4000 DNA 25-mers were successfully entrapped in the liposome, and minimal leakage was observed over the course of 8 months. When used in the sandwich immunoassay, a limit of detection of 4.1 ng/mL protective antigen was observed with an upper limit of 5000 ng/mL. Due to the endless combination of DNA oligonucleotide sequences, this assay lends itself perfectly for multiplexing on the order of tens to hundreds of analytes.     

Covalently Linked Perylene Diimide–Polydiacetylene Nanofibers Display Enhanced Stability and Photocurrent with Reversible FRET Phenomenon

Because of their unique structural and optical properties, 1D perylene diimide (PDI) derivatives have gained attention for use in optoelectronic devices. However, PDI‐containing self‐assembled supramolecular systems often are of limited use because they have supramolecular architectures that are held together by weak noncovalent π–π stacking, hydrogen bonding, and hydrophobic interactions. As a result, they are intrinsically unstable under solution‐processing conditions. To overcome this limitation, a polydiacetylene (PDA)‐based strategy is developed to construct a solvent‐resistant and stable PDI assembly. For this purpose, first the monomer PDI–BisDA is generated, in which two polymerizable diacetylene (DA) units are covalently linked to a PDI core. Importantly, 254 nm UV irradiation of self‐assembled PDI–BisDA nanofibers forms solvent‐resistant and stable PDI–PDA fibers. Owing to the presence of PDA, the generated polymer fibers display an increased photocurrent. In addition, the existence of PDA and PDI moieties in the fiber leads to the occurrence of switchable on–off fluorescence resonance energy transfer (FRET) between the PDI and reversibly thermochromic PDA chromophores.     

Detection of six single-nucleotide polymorphisms associated with rheumatoid arthritis by a loop-mediated isothermal amplification method and an electrochemical DNA chip

An electrochemical DNA chip using an electrochemically active intercalator and DNA probe immobilized on a gold electrode has been developed for genetic analysis. In this study, the six polymorphisms associated with rheumatoid arthritis (RA), N-acetyltransferase2 (NAT2) gene polymorphisms T341C, G590A, and G857A, methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms C677T and A1298C, and serum amyloid A1 (SAA1) gene promoter polymorphism C-13T were simultaneously detected by the electrochemical DNA chip and the loop-mediated isothermal amplification (LAMP) method, which is a novel technique for DNA amplification. Human genomic DNAs were extracted from blood, and the targets containing the six polymorphisms were amplified by the LAMP method. A sample containing the six LAMP products was reacted with the electrochemical DNA chip using a DNA detection system that controls hybridization reaction, washing, electrochemical detection, and data analysis automatically. A total of 31 samples were genotyped by this method, and the results were completely consistent with those determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis or the PCR direct sequence analysis. The time required for this method was only 2 h, and operations were very simple. Therefore, this method is expected to contribute to personalized medicine based on genotype.     

Screening for enzyme inhibitors by surface plasmon resonance combined with mass spectrometry

We have developed a novel strategy to identify enzyme inhibitors that interact directly with their enzyme targets. In the approach, an enzyme is immobilized on a sensor chip, and it is determined whether the immobilized enzyme is still active by incubation with model substrates and mass spectrometric analysis of the products. Putative inhibitors or mixtures containing putative inhibitors are then injected over the sensor chip for binding analysis with surface plasmon resonance. It is then tested whether the bound compounds inhibit the enzymatic activity by subsequent incubation with the model substrate and mass spectrometric analysis. If the bound compound inhibits the enzyme, the inhibitor is eluted from the enzyme and characterized by mass spectrometry. To test the strategy, it has been applied to the well-characterized interaction between trypsin and pure bovine pancreas trypsin inhibitor. Furthermore, fractions of plant extracts were screened for binding to and inhibition of carboxypeptidase B.     

The effect of laser radiation on the conductivity of poly(diacetylene)

It is demonstrated that an increase in the dark conductivity of poly(diacetylene) (PDA) upon laser irradiation in oxygen-containing media is determined by the formation of thin conducting surface layers. Pulsed laser irradiation at an energy of 0.04 J, a wavelength of 530 nm, and a pulse duration of 20 ns increases the conductivity of the surface PDA layers by four orders of magnitude. It is supposed that the laser-induced degradation and the subsequent stabilization of the surface PDA layers are mainly determined by photooxidation.     

Polydopamine/liposome coatings and their interaction with myoblast cells

Surface-mediated drug delivery is a recent concept, where active surface coatings are employed to deliver therapeutic cargo to cells. Herein, we explore the potential of liposomes embedded in polydopamine (PDA) coatings to serve as drug deposits stored on planar substrates. We quantify the PDA growth rate on glass by XPS and show that PDA coatings support myoblast adherence and proliferation. Further, PDA capping layers were deposited on glass substrates precoated with poly(L-lysine) and zwitterionic liposomes. Already thin PDA capping layers render liposome coated surfaces cell adhesive. We experimentally show for the first time, the internalization of a model hydrophobic cargo, that is, fluorescent lipids embedded within the lipid bilayer of liposomes by the cells from the surface. This is evident from the fluorescence exhibited by the cells grown on PDA coatings containing fluorescently labeled liposomes, with the highest fluorescent intensity found in the close proximity of the cell nuclei. The cargo uptake efficiency depends on the thickness of the PDA capping layer and the cell residence time on the coated substrates. Taken together, we demonstrate the first step toward the establishment of a versatile approach using liposomal drug deposits in polymer thin films for surface-mediated drug delivery.     

Micro-contact printing of polydiacetylene liposomes using hydrophilic stamps

Micron-sized polydiacetylene (PDA) liposome patterns have been fabricated on titanium (Ti) substrates using a micro-contact printing (micro-CP) technique. Two types of stamps (PDMS and agarose) and inking methods ("soaking" and "dropping") are used for micro-CP, and we compare their effect on the morphology of the PDA patterns. The size and morphology of the patterned PDA liposomes are analysized by optical and fluorescence microscopies and atomic force microscopy (AFM). When the agarose stamp is inked by the "dropping" method, PDA patterns are most efficiently transferred to the Ti substrate. However, the thickness of the transferred PDA patterns is not homogeneous, with the edge of the transferred pattern being thicker than its center. In contrast, when the PDMS stamp is used for micro-CP, the center of the pattern is thicker than the edge. Red fluorescence patterns are readily obtained by heat treatment of the PDA-immobilized solid substrate. The intensity of the fluorescence of the samples is consistent with the results of optical microscopy and AFM experiments.     

Chip-based analysis of protein-protein interactions by fluorescence detection and on-chip immunoprecipitation combined with microLC-MS/MS analysis

A new chip-based method to identify protein-protein interactions was developed using the guanine nucleotide exchange factor GRF2 and two interacting proteins, Ras and calmodulin, as model proteins. A generic immobilization strategy for FLAG-tagged bait proteins on a protein-repellent streptavidin chip surface was implemented by presentation of an oriented anti-FLAG antibody. A flow cell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with the two analytes was verified by fluorescence assays. On-chip tryptic digest assays were then performed on the capture surface and analyzed by microLC-MS/MS. The interaction of GRF2 with calmodulin and Ras was demonstrated, and the lower limit of detection was determined. We also implemented an on-chip immunoprecipitation assay to identify GRF2-binding partners from complex protein mixtures. Cells overexpressing FLAG-GRF2 were lysed and then incubated with the anti-FLAG chip. In addition to detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identify endogenous levels of proteins, bound to recombinant bait proteins. This chip-based method has the advantage that no subsequent gel separations of protein complexes prior to LC-MS analysis are required and is therefore amenable to miniaturized high-throughput determination of protein-protein interactions.     

Characterization of different sequences of DNA on si substrate by atomic force microscopy and gold nanoparticle labeling

In this paper, different sequences of single-strand DNA modified on Si substrate were studied taking advantages of the high resolution of atomic force microscopy (AFM) and signal enhancement of gold nanoparticles. Two sequences of single-strand DNA, as a model, were immobilized on Si substrate and hybridized with their sequence-complementary DNA molecules modified respectively with two sizes of gold nanoparticles. The surface of Si substrate was characterized through detecting the size and coverage of gold nanoparticles by AFM. Results demonstrated that different sizes of gold nanoparticles represented different sequences of DNA immobilized on the substrate. Density and distribution of DNA on Si substrate can be investigated by AFM imaging using gold nanoparticles as topographic markers. Compared to other sensitive methods such as fluorescence energy transfer, X-ray photoelectron, and radiolabeling experiments, this approach is advantageous in terms of high spatial resolution in sub-micrometer scale. This new method will be beneficial in the characterization of DNA immobilized on chip surfaces.     

Inkjet printing of conjugated polymer precursors on paper substrates for colorimetric sensing and flexible electrothermochromic display

Inkjet-printable aqueous suspensions of conjugated polymer precursors are developed for fabrication of patterned color images on paper substrates. Printing of a diacetylene (DA)-surfactant composite ink on unmodified paper and photopaper, as well as on a banknote, enables generation of latent images that are transformed to blue-colored polydiacetylene (PDA) structures by UV irradiation. Both irreversible and reversible thermochromism with the PDA printed images are demonstrated and applied to flexible and disposable sensors and to displays.     

Dynamic DNA hybridization on a chip using paramagnetic beads

Dynamic DNA hybridization is presented as an approach to perform gene expression analysis. The method is advantageous because of its dynamic supplies of both DNA samples and probes. The approach was demonstrated on a microfluidic platform by incorporating paramagnetic beads as a transportable solid support. A glass chip was fabricated to allow simultaneous interrogation of eight DNA target samples by DNA probes. DNA targets were immobilized on beads via streptavidin-biotin conjugation or base pairing between oligonucleotide residues. The DNA/bead complex was introduced into the device in which hybridization took place with a complementary probe. The hybridized probe was then removed by heat denaturation to allow the DNA sample to be interrogated again by another probe with a different sequence of interest. A pneumatic pumping apparatus was constructed to transport DNA probes and other reagents into the microfluidic device while hydrostatic pumping was used for the introduction of paramagnetic beads with samples. After investigating three types of paramagnetic beads, we found Dynabeads Oligo(dT)25 best suited this application. Targets on the beads could be sequentially interrogated by probes for 12 times, and the hybridization signal was maintained within experimental variation. Demonstration of specific hybridization reactions in an array format was achieved using four synthesized DNA targets in duplicate and five probes in sequence, indicating the potential application of this approach to gene expression analysis.     

Detection of Cryptosporidium parvum using oligonucleotide-tagged liposomes in a competitive assay format

To meet the technical challenge of accurately and rapidly detecting Cryptosporidium parvum oocysts in environmental water, the authors developed a single-use visual-strip assay. The first step in the overall assay procedure involves extracting C. parvum's mRNA coding for heat-shock protein hsp70, followed by amplification using nucleic acid sequence-based amplification (NASBA) methodology as described previously (Baeumner, A. J.; Humiston, M.; Montagna, R. A.; Durst, R. A. Anal. Chem., in press). Subsequently, generated amplicons are hybridized with dye-entrapping liposomes bearing DNA oligonucleotides (reporter probes) and biotin on their surface. The liposome-amplicon complex is then allowed to migrate upward on a nitrocellulose membrane strip. On the nitrocellulose strip, antisense-reporter probes are immobilized in a capture zone and antibiotin antibodies are immobilized in a second zone above the capture zone. Depending on the presence or absence of amplicon in the sample, the liposomes will bind to the capture zone, or they will be caught via their biotin tag in the second zone. Visual detection or gray-scale densitometry allows the quantification of liposomes that are present in either zone. The detection limit of the assay was determined to be 80 fmol amplicon/test. High accuracy and an internal assay control is established using this competitive format, because the presence or absence of liposomes can be quantified in the two capture zones.     

利用微型生物光学传感器的竞争免疫检测法检测牛奶中氨苄青霉素

利用微型表面等离子体共振的生物传感器测量了残留在牛奶中的氨苄青霉素的浓度.采用竞争抑制试验的方法,即先将定量的单克隆抗氨苄青霉素抗体（3H295）和含氨苄青霉素的牛奶样品混合,样品中氨苄青霉素即与抗体结合,然后将该混合样品通入传感器表面,传感器的表面共价固定了氨苄青霉素分子,通过生物特异相互作用分析,检测样品中剩余的抗体,从而得到氨苄青霉素的浓度.样品的测量时间为10min,最低检测限为2.5 ng/mL,低于欧盟标准4 ng/mL.该检测方法的测量时间短、重复性好,批间测量的变异系数为5.4%,表明该方法能满足实际测量要求.     

Electrokinetically controlled DNA hybridization microfluidic chip enabling rapid target analysis

Biosensors and more specifically biochips exploit the interactions between a target analyte and an immobilized biological recognition element to produce a measurable signal. Systems based on surface nucleic acid hybridization, such as microarrays, are particularly attractive due to the high degree of selectivity in the binding interactions. One of the drawbacks of this reaction is the relatively long time required for complete hybridization to occur, which is often the result of diffusion-limited reaction kinetics. In this work, an electrokinetically controlled DNA hybridization microfluidic chip will be introduced. The electrokinetic delivery technique provides the ability to dispense controlled samples of nanoliter volumes directly to the hybridization array (thereby increasing the reaction rate) and rapidly remove nonspecific adsorption, enabling the hybridization, washing, and scanning procedures to be conducted simultaneously. The result is that all processes from sample dispensing to hybridization detection can be completed in as little as 5 min. The chip also demonstrates an efficient hybridization scheme in which the probe saturation level is reached very rapidly as the targets are transported over the immobilized probe site enabling quantitative analysis of the sample concentration. Detection levels as low as 50 pM have been recorded using an epifluorescence microscope.     

Liposome Crosslinked Polyacrylamide/DNA Hydrogel: a Smart Controlled‐Release System for Small Molecular Payloads

A novel stimuli‐responsive hydrogel system with liposomes serving as both noncovalent crosslinkers and functional small molecules carriers for controlled‐release is developed. Liposomes can crosslink polyacrylamide copolymers functionalized with cholesterol‐modified DNA motifs to yield a DNA hydrogel system, due to the hydrophobic interaction between cholesteryl groups and the lipid bilayer of liposomes. Functional information encoded DNA motifs on the polymer backbones endow the hydrogel with programmable smart responsive properties. In a model system, the hydrogel exhibits stimuli‐responsive gel‐to‐sol transformation triggered by the opening of DNA motifs upon the presence of a restriction endonuclease enzyme, EcoR I, or temperature change, realizing the controlled‐release of liposomes which are highly efficient carriers of active small molecules payloads. Two active molecules, 1,1‐dioctadecyl‐3,3,3,3‐tetramethylindodicarbocyanine perchlorate (DiIC18(5)) and calcein, are chosen as the hydrophobic and hydrophilic model payloads, respectively, to address the feasibility of the releasing strategy. Moreover, the hydrogel exhibits injectable property as well as self‐recovery behaviors.     

Visualization and Quantitative Detection of Friction Force by Self‐Organized Organic Layered Composites

Visualization and quantitative detection of external stimuli are significant challenges in materials science. Quantitative detection of friction force, a mechanical stress, is not easily achieved using conventional stimuli‐responsive materials. Here, the quantitative detection of friction force is reported, such as the strength and accumulated ammount, from the visible color of organic layered composites consisting of polydiacetylene (PDA) and organic amines without an excitation light source. The composites of the layered diacetylene monomer crystal and interlayer organic amine are synthesized through self‐organization from the precursor solution. After topochemical polymerization, the layered composites based on PDA show tunable temperature‐responsive and mechanoresponsive color‐change properties depending on the types of interlayer amines. The layered composites are homogeneously coated on a filter paper. The change in color of the paper is quantitatively used to visualize the strength and accumulated amount of the applied friction force. Furthermore, writing pressure is measured by friction force using the paper device.     

Aptamer biosensors for label-free colorimetric detection of human IgE based on polydiacetylene (PDA) supramolecules

In this study, we demonstrate an aptamer-based biosensor (apta-biosensor) using PDA liposomes for label-free detection of allergy diagnosis by hIgE detection. In order to detect the target hIgE, the surface of PDA liposome were functionalized with hIgE antibody and anti-hIgE aptamer as a receptor, and the target hIgE onto the receptors was detected by the change of fluorescence signal. The hIgE antibody-modified PDA liposome biosensor had a serious problem that the immune reaction between receptor and target could not powerfully affect the change of florescence signal on PDA liposome. In order to solve this problem, the anti-hIgE aptamer which was far smaller than whole antibody was introduced as the receptor for the PDA liposome system. An aptamer-based PDA liposome biosensor was able to measure a quantity of target protein with various concentrations and at this time the detection limit was 141 ng/mL of the hIgE concentration. These results enabled diagnosis of allergy disease by an aptamer-based PDA liposome biosensor because real allergic patients showed high concentration of hIgE in serum (greater than 290 ng/mL). Therefore, we suggest that aptamer-modified PDA supramolecules as promising candidates for development of label-free colorimetric biosensors.     

Ganglioside-liposome immunoassay for the ultrasensitive detection of cholera toxin

An extremely sensitive bioassay has been developed for cholera toxin (CT) detection, using ganglioside-incorporated liposomes. Cholera is a diarrheal disease, often associated with water or seafood contamination. Ganglioside GM1 was used to prepare the liposomes by spontaneous insertion into the phospholipid bilayer. CT recognition and signal generation is based on the strong and specific interaction between GM1 and CT. In a sandwich immunoassay, CT was detected as a colored band on the nitrocellulose membrane strip, where CT bound to GM1-liposomes can be captured by immobilized antibodies. The intensity of the band could be visually estimated or measured by densitometry, using computer software. The limit of detection (LOD) of CT in the assay system was found to be 10 fg/mL which is equivalent to 8 zmol in the 70-microL sample. The assay was also tested with water samples spiked with CT, providing a LOD of 0.1-30 pg/mL, which is much better than previously reported limits of detection from other assays. The assay could be completed within 20 min. These results demonstrate that the bioassay developed for CT is rapid and ultrasensitive, suggesting the possibility for detecting CT, simply and reliably, in field screening.     

Selection of ligands for affinity chromatography using quartz crystal biosensor

This paper described a new strategy for rapid selecting ligands for application in affinity chromatography using a quartz crystal microbalance (QCM) biosensor. An aminoglycoside antibiotic drug, kanamycin (KM), was immobilized on the gold electrodes of the QCM sensor chip. The binding interactions of the immobilized KM with various proteins in solution were monitored as the variations of the resonant frequency of the modified sensor. Such a rapid screen analysis of interactions indicated clearly that KM-immobilized sensor showed strong specific interaction only with lysozyme (LZM). The resultant sensorgrams were rapidly analyzed by using a kinetic analysis software based on a genetic algorithm to derive both the kinetic rate constants (k(ass) and k(diss)) and equilibrium dissociation constants (K(D)) for LZM-KM interactions. The immobilized KM showed higher affinity to LZM with a dissociation constant on the order of 10(-5) M, which is within the range of 10(-4)-10(-8) M and suitable for an affinity ligand. Therefore, KM was demonstrated for the first time as a novel affinity ligand for purification of LZM and immobilized onto the epoxy-activated silica in the presence of a high potassium phosphate concentration. The KM immobilized affinity column has proved useful for a very convenient purification of LZM from chicken egg white. The purity of LZM obtained was higher than 90%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction. These results confirmed that the selected KM ligand is indeed a valuable affinity ligand for purification of LZM. The new screening strategy based on a QCM biosensor is expected to be a promising way for rapid selecting specific ligands for purifying other valuable proteins.