Recovery Of Fluorescence Intensity Research Articles

A Near‐IR Fluorimetric Chemosensing System for the Turn‐On Detection of Hydrogen Sulfate Anion

AbstractWe report a near‐IR fluorescence turn‐on chemosensing system for the selective detection of HSO4− anion in aqueous media. This system utilizes the equilibrium between the fluorescent pyrylium salt and its non‐fluorescent diketone form. The pyrylium compound we synthesized (λflo=674 nm) rapidly converts to its diketone form in the aqueous medium (ethanol‐water 4 : 1). HSO4− anion provides proton to diketone form inducing its conversion to pyrylium. Therefore, the sensor system responds to the presence of HSO4− by an increase in fluorescence. The change in the fluorescence increase is linear within the 20–55 μM range with a limit of detection of 12.8 μM. The system demonstrates high selectivity for HSO4− over other anions, as validated through fluorimetric titration studies. Additionally, interference studies highlight the minimal impact of common anions on bisulfate detection. Reversibility tests reveal the pronounced reversibility of the sensor system, with over 90 % recovery of fluorescence intensity achieved in subsequent iterations.

A “turn-on” approach for rapid detection of tetracycline based on supramolecular aggregates of water soluble perylene diimide

Antibiotic residue in animal foods has aroused great attention. Herein, we report a “turn-on” approach via supramolecular aggregates of water soluble perylene diimide (PDI) for rapid detection of tetracycline (TC), which is very important to food quality and safety. The disassembly of PDI aggregate was promoted owing to the coordination of Eu3+ and TC, leading to a significant recovery of fluorescence intensity. The sensing mechanism and performance of this approach were studied by absorption and emission spectra. The limit of detection (LOD) and the operation range were 12.11 nM and 0.1–30 μM, respectively. This method features simple acquisition, free of organic solvents, rapid detection, high selectivity and sensitivity, ease of observation and operation. It could be applied to detecting TC in Chicken, Fish and Pork with good recovery. To our knowledge, this is the first report of TC based on PDI probe in aqueous media.

A "turn-on" fluorescence resonance energy transfer aptasensor based on carbon dots and gold nanoparticles for 17β-estradiol detection in sea salt.

17β-estradiol is abused in the food industry. Excess 17β-estradiol can disturb the endocrine system or cause many diseases including obesity, diabetes, cardiac-cerebral vascular disease, and cancers in the human body. A "turn-on" fluorescence resonance energy transfer (FRET) aptasensor based on carbon dots (CDs) and gold nanoparticles (AuNPs) was developed for the detection of 17β-estradiol. A thiol-modified oligonucleotide was conjugated to AuNPs and amino modified oligonucleotide was linked to CDs. The 17β-estradiol aptamer was hybridized with the two oligonucleotides, shortening the distance between CDs and AuNPs. With 360 nm UV light excitation, FRET occurred between CDs and AuNPs. The system was "turn-off". When 17β-estradiol was detected, the aptamer specifically bound to 17β-estradiol, and the FRET system was destroyed, leading to the "turn-on" phenomenon. The fluorescence intensity recovery was detected in the concentration range of 400 pM to 5.5 μM. The limit of detection (LOD) was 245 pM. The FRET aptasensor demonstrated good selectivity for 17β-estradiol detection. Reasonable spiked recoveries were obtained in sea salt samples. It showed the potential for estrogen detection in food safety and environmental applications.

A turn‐on fluorescence assay for heparin based on DNA‐templated gold nanoclusters via ET

AbstractA novel fluorescence turn‐on assay for heparin has been developed based on DNA‐templated gold nanoclusters (AuNCs) and Cytochrome c (Cyt c). In the design, poly A (A15) ssDNA was selected as a template for fabricating AuNCs. AuNCs possess strong fluorescence emission at 471 nm. Upon addition of Cyt c, the fluorescence of AuNCs was quenched effectively by electron transfer (ET) between AuNCs and the heme cofactor of Cyt c. In the presence of heparin, the much higher electrostatic binding of heparin to Cyt c caused Cyt c to be away from AuNCs, leading to a recovery of fluorescence intensity of AuNCs. The proposed method was found to be simple, fast, and sensitive for heparin detection, with a linear range from 0.5 μg/mL to 12 μg/mL, and a detection limit of 0.15 μg/mL. Furthermore, this work was successfully applied to detection of heparin in fetal bovine serum samples, with good recoveries from 93.53% to 103.6%.

Sensor array for rapid pathogens identification fabricated with peptide-conjugated 2D metal-organic framework nanosheets

Rapid and efficient identification of pathogen is indispensable in clinical diagnosis because of the extremely severe consequence induced by pathogens infection. Taking advantages of the various compositions of pathogen surfaces, in this work we have proposed a simple and rapid sensor array for different pathogens identification and discrimination based on two-dimensional metal–organic frameworks (2D-MOFs) conjugated with functional peptide. Owing to the outstanding physical and chemical properties, ultrathin 2D-MOFs can adsorb dye-labeled peptides and consequently quench the fluorescence quickly and efficiently. Nevertheless, in the presence of pathogens, the anionic surface and different bimolecular composition of pathogens will endow them to interact with the 2D-MOFs or peptides through the diverse non-specific interactions. Subsequently, the dye-labeled peptides can be released with a recovery of fluorescence intensity. Consequently, the sensor array can identify various pathogens rapidly and efficiently based on the distinct fluorescence response patterns. Moreover, experimental results reveal that this approach can discriminate different pathogens with 100% accuracy, even in urine or mixed samples, demonstrating its reliability and effectiveness in the complex biological samples. More importantly, the procedure of pathogens identification by using this sensor array is very fast with only ~ 15 min, indicating the great potential applications in the timely clinical treatments and reliable monitoring of infectious disease trends.

Bipyridine- and Copper-Functionalized N-doped Carbon Dots for Fluorescence Turn Off-On Detection of Ciprofloxacin.

Herein, a fluorescence turn off-on nanosensor has been successfully developed using functionalized N-doped carbon dots (N-CDs) as the label-free sensing probe for the ultrasensitive detection of Cu2+ ions first and then ciprofloxacin (CIP), one of the most commonly used antibiotics for disease control, in the presence of bipyridine. The homogeneous and narrowly distributed N-CDs with a mean size of 5.7 nm and a high quantum yield of 84% are fabricated via the hydrothermal method in the presence of citric acid and ethylenediamine as the carbon and nitrogen sources, respectively. The Cu2+ ions serve as both analyte and fluorescence quenchers in the sensing platform of N-CDs, and a good linear response to Cu2+ in the range of 0.01-0.35 μM with a limit of detection (LOD) of 0.076 nM is observed. Then, 0.35 μM Cu2+ is used as the fluorescence quencher of N-CDs to build up the fluorescence turn off-on sensing probe for the detection of CIP using bipyridine (bipy) as the linker for CIP and Cu2+ ions. The addition of CIP to the bipy-Cu@N-CD composites triggers the formation of CIP-bipy-Cu conjugate as well as the release of N-CDs, resulting in the recovery of fluorescence intensity after 6 min of incubation. The sensing probe exhibits a two-phase linear response to CIP in the concentration range of 0.05-1 and 1-50 μM with a LOD of 0.4 nM. In addition, the bipy-Cu@N-CD probe shows high sensitivity toward CIP over the 19 other interferences. Good recovery of 96-110% is also observed when 0.1-0.9 μM CIP is spiked into the real samples. Results obtained in this study clearly demonstrate a newly developed sensing platform with rapid detection of metal ions and antibiotics, which can open an avenue to develop highly efficient and robust sensing probes for the detection of metal ions, organic metabolites, and biomarkers in biological applications.

Raster adaptive optics for video rate aberration correction and large FOV multiphoton imaging.

Removal of complex aberrations at millisecond time scales over millimeters in distance in multiphoton laser scanning microscopy limits the total spatiotemporal imaging throughput for deep tissue imaging. Using a single low resolution deformable mirror and time multiplexing (TM) adaptive optics, we demonstrate video rate aberration correction (5 ms update rate for a single wavefront mask) for a complex heterogeneous distribution of refractive index differences through a depth of up to 1.1 mm and an extended imaging FOV of up to 0.8 mm, with up to 167% recovery of fluorescence intensity 335 µm from the center of the FOV. The proposed approach, termed raster adaptive optics (RAO), integrates image-based aberration retrieval and video rate removal of arbitrarily defined regions of dominant, spatially varied wavefronts. The extended FOV was achieved by demonstrating rapid recovery of up to 50 distinct wavefront masks at 500 ms update rates that increased imaging throughput by 2.3-fold. Because RAO only requires a single deformable mirror with image-based aberration retrieval, it can be directly implemented on a standard laser scanning multiphoton microscope.

Fluorescence sensor for bovine serum albumin detection based on the aggregation and release of CdS QDs within CMC

Here, an efficient in vitro fluorescence biosensor for bovine serum albumin (BSA) based on the aggregation and release of CdS quantum dots (QDs) within carboxymethyl cellulose (CMC) was investigated and discussed. The aggregation of CdS QDs within CMC was prepared by electrostatic interaction. The release of CdS QDs was due to strong covalent linking between BSA and CMC, thus leading to the more favorable combination of BSA and CMC in the system with the presence of BSA. The detection of BSA was based on the quenching and recovery of fluorescence intensity in the system, which was caused by the aggregation and release of CdS QDs. An excellent linear relationship (R2 = 0.99286) was obtained between fluorescence intensity and BSA concentration (0.05–2.00 μM) with a detection limit of 10−8 M. In addition, the detection method showed high selectivity towards BSA and good stability. These results suggest that the method can be utilized as an efficient and highly selective reagent for quantification of BSA for in vitro biological science.

Tyrosinase-mediated Peptide Conjugation with Chitosan-coated Gold Nanoparticles.

The conjugation of biomolecules, such as protein, sugar, and DNA, with metal nanoparticles is an important technique for bioassay and biomaterial preparation. In this study, we aim to enzymatically immobilize a functional peptide on gold nanoparticles (AuNPs) using a single-step reaction. We used tyrosinase, a catechol oxidase, to immobilize an enzymatic peptide. We performed immobilization experiments of a fluorescent compound-linked caspase-3 substrate peptide using tyrosinase on chitosan-coated AuNPs. Peptides were effectively immobilized onto the AuNPs depending on the presence of tyrosine within the sequence, which suggests the DOPA-quinone produced from tyrosine, via tyrosinase, is connected to the chitosan amino group. Although fluorescent emission from the immobilized capase-3 substrate was quenched by AuNPs, fluorescence intensity recovery occurred due to the addition of caspase-3. Thus, we were able to easily prepare functional AuNPs that can be used for a caspase-3 activity assay. Our results indicate that the tyrosinase-mediated peptide link to chitosan-coated particles is a useful technique for preparing functionalized nanoparticles.

Assembly-enhanced fluorescence from metal nanoclusters and quantum dots for highly sensitive biosensing

Fluorescent nanoparticles exhibit unique optical properties for applications in imaging and sensing. Herein, the enhancement of luminescence emission from self-assembled nanoparticles including gold nanoclusters, cadmium sulphide and near-infrared silver selenide quantum dots are reported. Addition of zinc and pyrophosphate ions induces assembly and disassembly of the particle aggregates and the corresponding intensified and recovery of fluorescence intensity, respectively. The enhancement is due to assembly-enhanced emission of metal nanoclusters and passivation of the surface trap states of quantum dots. With the hydrolysis of pyrophosphate catalyzed by alkaline phosphatase, the released zinc ions from pyrophosphate-zinc ion complexes mediate nanoparticle assembly and lead to luminescence regeneration. The assembly-induced fluorescence enhancement can be applied for sensitive assay of alkaline phosphatase with a detection limit of 3.4 pM in a continuous and real-time way. This method is capable of screening enzyme inhibitor and analyzing biological samples.

Determination of diffusion coefficients in live cells using fluorescence recovery after photobleaching with wide-field fluorescence microscopy.

Fluorescence recovery after photobleaching (FRAP) enables characterization of quantitative dynamic properties such as diffusion coefficients of fluorescent molecules in living cells by analyzing the recovery of fluorescence intensity after photobleaching in a specific cellular compartment or area. To quantitatively determine high intracellular diffusion coefficients, a suitable optical system as well as an appropriate model for fast diffusion analysis is necessary. Here, we propose a procedure to quantify the diffusion coefficient of rapidly-diffusing fluorescent molecules that makes use of an epi-fluorescence microscope with a photobleaching laser in combination with established models for diffusion analysis. Analysis for the diffusion coefficients of tandemly oligomerized green flurescent proteins (GFPs) in living cells when changing the photobleaching times showed that photobleaching with shorter times than the diffusion speed indicated not the only way to obtain appropriate diffusion coefficients of fast-moving molecules. Our results also showed that the apparent spreading of the effective radius of the photobleached area works as a correction factor for determining appropriate diffusion coefficients of fast-moving molecules like monomeric GFPs. Our procedure provides a useful approach for quantitative measurement of diffusion coefficients in living cells. This procedure is relevant for characterizing dynamic molecular interactions, especially of fast-moving molecules, and is relevant for studies in many biological fields.

N-Doped Graphene Quantum Dots-Decorated V2O5 Nanosheet for Fluorescence Turn Off-On Detection of Cysteine.

The development of a fast-response sensing technique for detection of cysteine can provide an analytical platform for prescreening of disease. Herein, we have developed a fluorescence turn off-on fluorescence sensing platform by combining nitrogen-doped graphene quantum dots (N-GQDs) with V2O5 nanosheets for the sensitive and selective detection of cysteine in human serum samples. V2O5 nanosheets with 2-4 layers are successfully synthesized via a simple and scalable liquid exfoliation method and then deposited with 2-8 nm of N-GQDs as the fluorescence turn off-on nanoprobe for effective detection of cysteine in human serum samples. The V2O5 nanosheets serve as both fluorescence quencher and cysteine recognizer in the sensing platform. The fluorescence intensity of N-GQDs with quantum yield of 0.34 can be quenched after attachment onto V2O5 nanosheets. The addition of cysteine triggers the reduction of V2O5 to V4+ as well as the release of N-GQDs within 4 min, resulting in the recovery of fluorescence intensity for the turn off-on detection of cysteine. The sensing platform exhibits a two-stage linear response to cysteine in the concentration range of 0.1-15 and 15-125 μM at pH 6.5, and the limit of detection is 50 nM. The fluorescence response of N-GQD@V2O5 exhibits high selectivity toward cysteine over other 22 electrolytes and biomolecules. Moreover, this promising platform is successfully applied in detection of cysteine in human serum samples with excellent recovery of (95 ± 3.8) - (108 ± 2.4)%. These results clearly demonstrate a newly developed redox reaction-based nanosensing platform using N-GQD@V2O5 nanocomposites as the sensing probe for cysteine-associated disease monitoring and diagnosis in biomedical applications, which can open an avenue for the development of high performance and robust sensing probes to detect organic metabolites.

Dual-Stimuli-Responsive Fluorescent Supramolecular Polymer Based on a Diselenium-Bridged Pillar[5]arene Dimer and an AIE-Active Tetraphenylethylene Guest

We report a new strategy to construct a multi-stimuli-responsive fluorescent supramolecular polymer by the strong host–guest interactions between a diselenium bond-bridged pillar[5]arene dimer and an AIE-active tetraphenylethylene (TPE)-containing neutral guest bearing two imidazole terminal binding sites. The resulting supramolecular polymer shows a remarkable fluorescence emission decrease at low concentration. Significantly, the diselenium bonds introduced into the supramolecular polymer serve as redox-responsive building blocks. Upon addition of reductants, the supramolecular polymer depolymerized owing to the cleavage of the covalent diselenium bonds in the system. On the other hand, competitive guests such as adiponitrile, which bind strongly with pillar[5]arenes, could lead the disassembly of the polymer to oligomers without breaking any covalent bonds in the system. These two types of depolymerization approaches can both result in the fluorescence intensity recovery of the system to a certain exte...

Real-time monitoring of microdistribution of antibody-photon absorber conjugates during photoimmunotherapy in vivo

Photoimmunotherapy (PIT) is an emerging low side effect cancer therapy based on a monoclonal antibody (mAb) conjugated with a near-infrared (NIR) phthalocyanine dye IRDye 700DX. IR700 is fluorescent, can be used as an imaging agent, and also is phototoxic. It induces rapid cell death after exposure to NIR light. PIT induces highly selective cancer cell death, while leaving most of tumor blood vessels unharmed, leading to an effect called super-enhanced permeability and retention (SUPR). SUPR significantly improves the effectiveness of the anticancer drug. Currently, the therapeutic effects of PIT are monitored using the IR700 fluorescent signal based on macroscopic fluorescence reflectance imagery. This technique, however, lacks the resolution and depth information to reveal the intratumor heterogeneity of mAb-IR700 distribution. We applied a minimally invasive two-channel fluorescence fiber imaging system by combining the traditional fluorescence imaging microscope with two imaging fiber bundles (~0.85mm). This method monitored mAb-IR700 distribution and therapeutic effects during PIT at different intratumor locations (e.g., tumor surface vs. deep tumor) in situ and in real time simultaneously. This enabled evaluation of the therapeutic effects in vivo and treatment regimens. The average IR700 fluorescence intensity recovery after PIT to the tumor surface is 91.50%, while it is 100.63% in deep tumors. To verify the results, two-photon microscopy combined with a microprism was also used to record the mAb-IR700 distribution and fluorescence intensity of green fluorescent protein (GFP) at different tumor depths during PIT. After PIT treatment, there was significantly higher IR700 fluorescence recovery in deep tumor than in the tumor surface. This phenomenon can be explained by increased vascular permeability immediately after NIR-PIT. Fluorescence intensity of GFP at the tumor surface decreased significantly more compared to that of deep tumor and in controls (no PIT).

Acid-Sensitive BODIPY Dyes: Synthesis through Pd-Catalyzed Direct C(sp3 )-H Arylation and Photophysics.

A novel palladium-catalyzed direct C(sp3 )-H arylation of the methyl group at the 8-position of BODIPY by bromoarenes was established. A deprotonative cross-coupling process was supposed to be involved in the reaction. This approach allowed us to attach electron-donating/withdrawing, halogen substituted aryls and a heteroaryl with a yield running from 55 to 99 %. Novel pH sensors, which in the absence of acid showed the occurrence of photoinduced electron transfer, were synthesized by attaching dimethylaniline to the methyl at the C8-position of BODIPY. The reference compounds with dimethylaniline directly attached to the C8-position were also synthesized and besides photoinduced electron transfer also showed a charge-transfer emission. Their photophysical properties were investigated by steady-state fluorescence, time-correlated single-photon counting and femtosecond fluorescence up-conversion. Time-dependent density functional (TD-DFT) electronic-structure calculations on the properties of the excited states corresponding to local excitation of the BODIPY core and to charge transfer were conducted. Upon addition of trifluoroacetic acid in toluene and ethanol, the partial fluorescence intensity recovery was at least an order of magnitude more efficient with the newly synthesized sensors compared to the traditional reference sensors. The improved sensitivity of these novel BODIPY-based pH sensors was attributed to less efficient proton-coupled electron transfer of the protonated species.

Closed-form formulas vs. PDE based numerical solution for the FRAP data processing: Theoretical and practical comparison

Fluorescence recovery after photobleaching (FRAP) is a widely used method to analyze (usually using fluorescence microscopy) the mobility of either fluorescently tagged or autofluorescent (e.g., photosynthetic) proteins in living cells. The FRAP method resides in imaging the recovery of fluorescence intensity over time in a region of interest previously bleached by a high-intensity laser pulse. While the basic principles of FRAP are simple and the experimental setup is usually fixed, quantitative FRAP data analysis is not well developed. Different models and numerical procedures are used for the underlying model parameter estimation without knowledge of how robust the methods are, i.e., the parameter inference step is not currently well established.In this paper we rigorously formulate the inverse problem of model parameter estimation (including the sensitivity analysis), making possible the comparison of different FRAP parameter inference methods. Then, in a study on simulated data, we focus on how three different methods for inference influence the error in parameter estimation. We demonstrate both theoretically and empirically that our new method based on a solution of a general initial–boundary value problem for the Fick diffusion partial differential equation exhibits less bias and narrower confidence intervals of the estimated diffusion parameter, than two closed formula methods.

Fabrication of folic acid-sensitive gold nanoclusters for turn-on fluorescent imaging of overexpression of folate receptor in tumor cells

Based on the fluorescence quenching of folic acid-sensitive bovine serum albumin-directed gold nanoclusters (BSA-AuNCs) via folic acid-induced the change of environment around BSA-AuNCs, we have constructed a turn on fluorescence imaging of folate receptor overexpressed tumor cells. In this paper, the primary fluorescence intensity of BSA-AuNCs was quenched via self-assembly of folic acid onto BSA-AuNCs to produce negligible fluorescence background, the linear range of the method was 0.1–100μg/mL with the limit of detection (LOD) of 30ng/mL (S/N=3); In the presence of overexpression of folate receptor on the surface of tumor cells, the primary fluorescence intensity of BSA-AuNCs turned on by folic acid desorbing from BSA-AuNCs, the linear range of method was 0.12–2μg/mL with the LOD of 20ng/mL (S/N=3). Additionally, due to specific and high affinity of folic acid and folate receptor, the probe had high selectivity for folate receptor, other interferences hardly changed the fluorescence intensity of the probe. Moreover, the text for cytotoxicity implied that the probe had no toxicity for tumor cells. Consequently, using the fluorescence probe, satisfactory results for the turn on imaging of folate receptor overexpressed tumor cells were obtained.A novel turn-on and red fluorescent probe for folate receptor overexpressed tumor cells was developed based on the recovery of fluorescence intensity of folic acid-sensitive BSA-AuNCs.

‘Turn-on’ fluorescence assay for inorganic phosphate sensing

Phosphorus (in the form of phosphate) is the second most abundant mineral in the human body and is essential for normal body activities. Abnormal concentrations of phosphate can cause serious disorders and may be fatal if not treated. One of the drawback of current phosphate sensing method is its lack of sensitivity and specificity. This issue can be overcome by using photoinduced electron transfer (PET) based fluorescent on/off sensors. Semiconductor quantum dots (QDs) are used for the purpose of phosphate sensing because of their excellent size tunable optical properties (fluorescence and absorbance). Here we report the synthesis and characterization of thiol capped cadmium telluride (CdTe) QDs and their use in ‘turn on’ assay for inorganic phosphate sensing. The assay utilizes europium nitrate which forms complex with CdTe QDs (QD-Eu complex) and quenches fluorescence of QDs through photoinduced electron transfer (PET) process. Addition of phosphate solution results in recovery of fluorescence intensity of TGA capped CdTe QDs. Fluorescence recovery was quantified in terms of the concentration of inorganic phosphate. The fluorescence intensity versus Pi concentration curve fits to the sigmoid shape after optimization for the desired range of phosphate concentration. This ‘turn on’ sensing assay method is reliable, repeatable and might be useful for determination of physiological inorganic phosphate level.

Magnetic Separation-Assistant Fluorescence Resonance Energy Transfer Inhibition for Highly Sensitive Probing of Nucleolin.

For the widely used "off-on" fluorescence (or phosphorescence) resonance energy transfer (FRET or PRET) system, the separation of donors and acceptors species was vital for enhancing the sensitivity. To date, separation of free donors from FRET/PRET inhibition systems was somewhat not convenient, whereas separation of the target-induced far-between acceptors has hardly been reported yet. We presented here a novel magnetic separation-assistant fluorescence resonance energy transfer (MS-FRET) inhibition strategy for highly sensitive detection of nucleolin using Cy5.5-AS1411 as the donor and Fe3O4-polypyrrole core-shell (Fe3O4@PPY) nanoparticles as the NIR quenching acceptor. Due to hydrophobic interaction and π-π stacking of AS1411 and PPY, Cy5.5-AS1411 was bound onto the surface of Fe3O4@PPY, resulting in 90% of fluorescence quenching of Cy5.5-AS1411. Owing to the much stronger specific interaction of AS1411 and nucleolin, the presence of nucleolin could take Cy5.5-AS1411 apart from Fe3O4@PPY and restore the fluorescence of Cy5.5-AS1411. The superparamagnetism of Fe3O4@PPY enabled all separations and fluorescence measurements complete in the same quartz cell, and thus allowed the convenient but accurate comparison of the sensitivity and fluorescence recovery in the cases of separation or nonseparation. Compared to nonseparation FRET inhibition, the separation of free Cy5.5-AS1411 from Cy5.5-AS1411-Fe3O4@PPY solution (the first magnetic separation, MS-1) had as high as 25-fold enhancement of the sensitivity, whereas further separation of the nucleolin-inducing far-between Fe3O4@PPY from the FRET inhibition solution (the second magnetic separation, MS-2) could further enhance the sensitivity to 35-fold. Finally, the MS-FRET inhibition assay displayed the linear range of 0.625-27.5 μg L(-1) (8.1-359 pM) and detection limit of 0.04 μg L(-1) (0.05 pM) of nucleolin. The fluorescence intensity recovery (the percentage ratio of the final restoring fluorescence intensity to the quenched fluorescence intensity of Cy5.5-AS1411 solution by 0.09 g L(-1) Fe3O4@PPY) was enhanced from 36% (for nonseparation) to 56% (for two magnetic separations). This is the first accurate evaluation for the effect of separating donor/acceptor species on the FRET inhibition assay.

Mobile lipid bilayers on gold surfaces through structure-induced lipid vesicle rupture.

Forming fluid supported lipid bilayers (SLBs) on a gold surface can enable various lipid-membrane-associated biomolecular interactions to be investigated by several surface sensing techniques, such as surface plasmon resonance and scanning tunneling microscopy. However, forming fluid SLBs on a gold surface through lipid vesicle deposition continues to pose a challenge. In this study, we constructed nanograting structures on a gold surface to induce lipid vesicle rupture for forming a mobile layer of SLBs. Observations based on fluorescence recovery after photobleaching showed that SLBs on the prepared grating supports had some fluidity, while SLBs on the planar support had no fluidity. The anisotropic fluorescence intensity recovery shape changes observed in the SLBs on the grating support suggested that a second layer of SLBs partially formed on top of the first layer in contact with the gold surface and extended along the grating structure. Comparisons of the relative amounts of second bilayer and the fluorescence recovery fractions on supports with various grating edge densities suggested that the second layer formed at the edge regions and that the coverage ratio was directly proportional to the grating edge density. All of these results showed that the grating edges could serve as vesicle-rupture-inducing sites for the formation of a mobile second SLB on a gold surface. The formation of the second layer of SLBs at the edge regions but not in the flat regions enabled us to determine the second layer locations and provided us with an opportunity to pattern mobile lipid bilayers on gold surfaces by controlling the edge locations.