Single-Molecule Flow Platform for the Quantification of Biomolecules Attached to Single Nanoparticles.

We describe here a flow platform for quantifying the number of biomolecules on individual fluorescent nanoparticles. The platform combines line-confocal fluorescence detection with near nanoscale channels (1-2 µm in width and height) to achieve high single-molecule detection sensitivity and throughput. The number of biomolecules present on each nanoparticle was determined by deconvolving the fluorescence intensity distribution of single-nanoparticle-biomolecule complexes with the intensity distribution of single biomolecules. We demonstrate this approach by quantifying the number of streptavidins on individual semiconducting polymer dots (Pdots); streptavidin was rendered fluorescent using biotin-Alexa647. This flow platform has high-throughput (hundreds to thousands of nanoparticles detected per second) and requires minute amounts of sample (∼5 µL at a dilute concentration of 10 pM). This measurement method is an additional tool for characterizing synthetic or biological nanoparticles.

Optically Encoded Semiconducting Polymer Dots with Single-Wavelength Excitation for Barcoding and Tracking of Single Cells.

Multiplexed optical encoding is emerging as a powerful technique for high-throughput cellular analysis and molecular assays. Most of the developed optical barcodes, however, either suffer from large particle size or are incompatible with most commercial optical instruments. Here, a new type of nanoscale fluorescent barcode (Pdot barcodes) was prepared from semiconducting polymers. The Pdot barcodes possess the merits of small size (∼20 nm in diameter), narrow emission bands (full-width-at-half-maximum (fwhm) of 30-40 nm), three-color emissions (blue, green, and red) under single-wavelength excitation, a high brightness, good pH and thermal stability, and efficient cellular uptake. The Pdot barcodes were prepared using a three-color and six-intensity encoding strategy; for ratiometric readout of the barcodes, one of the colors might be used as an internal reference. We used the Pdot barcodes to label 20 sets of cancer cells and then distinguished and identified each set based on the Pdot barcodes using flow cytometry. We also monitored and tracked single cells labeled with different Pdot barcodes, even through rounds of cell division. These results suggest Pdot barcodes are strong candidates for discriminating different labeled cell and for long-term cell tracking.

Squaraine-based polymer dots with narrow, bright near-infrared fluorescence for biological applications.

This article describes the design and development of squaraine-based semiconducting polymer dots (Pdots) that show large Stokes shifts and narrow-band emissions in the near-infrared (NIR) region. Fluorescent copolymers containing fluorene and squaraine units were synthesized and used as precursors for preparing the Pdots, where exciton diffusion and likely through-bond energy transfer led to highly bright and narrow-band NIR emissions. The resulting Pdots exhibit the emission full width at half-maximum of ∼36 nm, which is ∼2 times narrower than those of inorganic quantum dots in the same wavelength region (∼66 nm for Qdot705). The squaraine-based Pdots show a high fluorescence quantum yield (QY) of 0.30 and a large Stokes shift of ∼340 nm. Single-particle analysis indicates that the average per-particle brightness of the Pdots is ∼6 times higher than that of Qdot705. We demonstrate bioconjugation of the squaraine Pdots and employ the Pdot bioconjugates in flow cytometry and cellular imaging applications. Our results suggest that the narrow bandwidth, high QY, and large Stokes shift are promising for multiplexed biological detections.

Single-chain semiconducting polymer dots.

This work describes the preparation and validation of single-chain semiconducting polymer dots (sPdots), which were generated using a method based on surface immobilization, washing, and cleavage. The sPdots have an ultrasmall size of ∼3.0 nm as determined by atomic force microscopy, a size that is consistent with the anticipated diameter calculated from the molecular weight of the single-chain semiconducting polymer. sPdots should find use in biology and medicine as a new class of fluorescent probes. The FRET assay this work presents is a simple and rapid test to ensure methods developed for preparing sPdot indeed produced single-chain Pdots as designed.

Percutaneous Treatment of Large Venous Malformations in the Oral and Maxillofacial Regions Using Electrochemical Therapy Combined With Pingyangmycin.

PURPOSE: The aim of the present study was to evaluate the therapeutic outcome of using electrochemical therapy (ECT) combined with a sclerosing agent, pingyangmycin (bleomycin A5 hydrochloride; PYM), for large (>3 cm in diameter) venous malformations (VMs) in the oral and maxillofacial regions. PATIENTS AND METHODS: Thirty-five patients (15 male and 20 female; age range, 10 to 69 yr; mean age, 32 yr) with large VMs in the oral and maxillofacial region were treated with a combination of ECT and PYM under general anesthesia in the authors' department from June 2012 through May 2014. The size of the lesions varied from 3 × 3 to 12 × 15 cm. A repeated course of ECT and PYM was administered for larger VMs. The therapeutic interval was 3 months for ECT and 2 to 4 weeks for PYM. The dose of PYM for patients was 8 mg each time, and the injection concentration of PYM was 1.6 mg/mL. Patients were followed for 6 to 36 months. Therapeutic results were evaluated by clinical examination and Doppler ultrasonography before and after treatment. RESULTS: Of the 35 patients, 29 (82.9%) received 1 ECT treatment, 5 (14.3%) received 2 ECT treatments, and 1 (2.8%) received 3 ECT treatments. The number of PYM injection sessions was 1 to 5 (average, 2.5 times). According to the therapeutic criteria, the clinical outcome was excellent in 22 patients (62.9%), good in 10 patients (28.6%), and fair in 3 patients (8.5%). All patients (100%) had local swelling postoperatively that lasted approximately 1 to 2 weeks. Two patients (5.7%) had fever. No skin necrosis or nerve damage was found. CONCLUSIONS: Percutaneous treatment using ECT and PYM was a straightforward, safe, and reliable treatment modality for large VMs.

Hybrid semiconducting polymer nanoparticles as polarization-sensitive fluorescent probes.

Much work has been done on collapsed chains of conjugated semiconducting polymers and their applications as fluorescent probes or sensors. On surfaces spin-coated with semiconducting polymers, excitation energy transfer along the polymer backbone can be used to quickly and efficiently funnel energy to chromophores with localized energy minima. If each chromophore is immobilized within its matrix, this can result in a large fluorescence anisotropy. Through nanoprecipitation of a matrix polymer blended at low mass ratios with short-chain, hydrophobic, fluorescent semiconducting polymers, we took advantage of this large fluorescence anisotropy to make polarization-sensitive nanoparticles (NPs). These NPs are small (~7 nm in diameter), exhibit a high quantum yield of 0.75, and are easily functionalized to bind to protein targets. Excitation of the NPs with polarized light on a wide-field fluorescence microscope enabled monitoring of both protein location and changes in protein orientation.

Semiconducting polymer dots doped with europium complexes showing ultranarrow emission and long luminescence lifetime for time-gated cellular imaging.

Bright dots: Semiconducting polymer dots (Pdots) doped with europium complexes possess line-like fluorescence emission, high quantum yield, and long fluorescence lifetime. The Pdots successfully labeled receptors on cells. The long fluorescence lifetime of the Pdots was used to distinguish them from other red fluorescence emitting nanoparticles, and improve the signal-to-noise ratio for time-gated cellular imaging. PVK=poly(9-vinylcarbazole).

High-Stretchability, Ultralow-Hysteresis ConductingPolymer Hydrogel Strain Sensors for Soft Machines.

Highly stretchable strain sensors based on conducting polymer hydrogel are rapidly emerging as a promising candidate toward diverse wearable skins and sensing devices for soft machines. However, due to the intrinsic limitations of low stretchability and large hysteresis, existing strain sensors cannot fully exploit their potential when used in wearable or robotic systems. Here, a conducting polymer hydrogel strain sensor exhibiting both ultimate strain (300%) and negligible hysteresis (<1.5%) is presented. This is achieved through a unique microphase semiseparated network design by compositing poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) nanofibers with poly(vinyl alcohol) (PVA) and facile fabrication by combining 3D printing and successive freeze-thawing. The overall superior performances of the strain sensor including stretchability, linearity, cyclic stability, and robustness against mechanical twisting and pressing are systematically characterized. The integration and application of such strain sensor with electronic skins are further demonstrated to measure various physiological signals, identify hand gestures, enable a soft gripper for objection recognition, and remote control of an industrial robot. This work may offer both promising conducting polymer hydrogels with enhanced sensing functionalities and technical platforms toward stretchable electronic skins and intelligent robotic systems.

Covalent bonding of YIGSR and RGD to PEDOT/PSS/MWCNT-COOH composite material to improve the neural interface.

The development of coating materials for neural interfaces has been a pursued to improve the electrical, mechanical and biological performances. For these goals, a bioactive coating was developed in this work featuring a poly(3,4-ethylenedioxythiophene) (PEDOT)/carbon nanotube (CNT) composite and covalently bonded YIGSR and RGD. Its biological effect and electrical characteristics were assessed in vivo on microwire arrays (MWA). The coated electrodes exhibited a significantly higher charge storage capacity (CSC) and lower electrochemical impedance at 1 kHz which are desired to improve the stimulating and recording performances, respectively. Acute neural recording experiments revealed that coated MWA possess a higher signal/noise ratio capturing spikes undetected by uncoated electrodes. Moreover, coated MWA possessed more active sites and single units, and the noise floor of coated electrodes was lower than that of uncoated electrodes. There is little information in the literature concerning the chronic performance of bioactively modified neural interfaces in vivo. Therefore in this work, chronic in vivo tests were conducted and the PEDOT/PSS/MWCNT-polypeptide coated arrays exhibited excellent performances with the highest mean maximal amplitude from day 4 to day 12 during which the acute response severely compromised the performance of the electrodes. In brief, we developed a simple method of covalently bonding YIGSR and RGD to a PEDOT/PSS/MWCNT-COOH composite improving both the biocompatibility and electrical performance of the neural interface. Our findings suggest that YIGSR and RGD modified PEDOT/PSS/MWCNT is a promising bioactivated composite coating for neural recording and stimulating.

Diurnal rhythm of free estradiol during the menstrual cycle.

OBJECTIVE: To investigate the diurnal rhythm of estrogens in normally cyclic women during reproductive life. DESIGN: Multiple saliva sampling in normally cyclic healthy women during reproductive life at different phases of their menstrual cycles was carried out. METHODS: Salivary estradiol was measured by radioimmunoassay in samples collected every 2 h for 24 h from 15 normally cyclic healthy women during reproductive life during the menstrual phase, the late follicular/peri-ovulation phase, the early to mid luteal phase and the late luteal phase, respectively, of their menstrual cycles. The levels of salivary estradiol were analyzed by means of periodic regression. RESULTS: A daily biological rhythm of free estradiol was found after quantification with a nonlinear periodic regression model. The observed diurnal free estradiol rhythm consists of two major components: an asymmetrically peaked diurnal cycle and ultradian harmonics in the range of 6 to 12 h. The diurnal and ultradian rhythms were remarkably consistent throughout the menstrual cycle in terms of mesor (24 h mean level), peak width and amplitude. There was a tendency for the 24-h rhythm acrophases to converge in the early morning, while the acrophase of the menstrual phase occurred significantly later than in the late follicular/peri-ovulation phase. CONCLUSIONS: The diurnal rhythm of estradiol has a similar complex temporal organization for different menstrual phases. The menstrual cycle mainly modulates the acrophase of the diurnal rhythm.

Influence of hydroxypropyl methylcellulose edible coating on fresh-keeping and storability of tomato.

The effect of application of cellulose-based edible coating, hydroxypropyl methylcellulose (HPMC) to mature-green tomatoes on the firmness and color was investigated. Tomatoes were stored at 20 degrees C for up to 18 days. Firmness decreased as storage time increased in all treatments. However, application of HPMC edible coating delayed softening of tomatoes during 18 days of storage at 20 degrees C. At days 7, 13 and 18, the firmness of tomatoes coated with HPMC was significantly (P < or = 0.05) greater than the firmness of uncoated tomatoes. The study also confirmed that HPMC coatings could significantly (P < or = 0.05) delay the changes in color of tomatoes stored at 20 degrees C. The ripening of tomatoes from the pink stage to the red stage was successfully retarded. HPMC coating could extend the shelf life of fresh tomatoes. The retardation of the rate of loss of firmness could reduce the economic loss that would result from spoilage by mechanical injury during transportation of tomatoes.

[Relationships between melatonin and cortisol and the status of disease in patients with bronchial asthma].

OBJECTIVE: The aim of the study was to elucidate the interaction and clinical significance between melatonin and cortisol and the status of disease in patients with bronchial asthma. METHODS: Ten mild persistent and 10 moderate-to-severe persistent asthma patients were recruited to participate in the study. Fifteen normal subjects served as contrds Salivary free melatonin and cortisol were measured simultaneously by radioimmunoassay in all subjects, and 8 salivary samples were collected in a series during a 24-hour period in each subject. The intensity of light was restricted to natural light at room during the daytime and less than 50 lux at night. RESULTS: The results showed that salivary free melatonin levels were significantly lower in mild (15.5 +/- 5.3) micro g/L and moderate-to-severe (7.1 +/- 2.5) micro g/L persistent asthma patients as compared to control subjects (28.9 +/- 8.7) micro g/L (F = 4.47, P < 0.05; F = 7.61, P < 0.01, respectively). The results also showed that salivary free cortisol levels were significantly lower in mild (3.1 +/- 0.5) micro g/L and moderate-to-severe (4.2 +/- 0.5) micro g/L persistent asthma patients as compared to control subjects (5.9 +/- 0.7) micro g/L (F = 10.45, P < 0.01; F = 5.21, P < 0.05, respectively). The amplitude of salivary free cortisol level was reduced in mild and moderate-to-severe persistent asthma patients, and salivary cortisol peak level was significantly delayed in mild and moderate-to-severe persistent asthma patients (P < 0.05, P < 0.01, respectively). There were no significant correlations between the salivary free melatonin and cortisol levels in control and mild persistent asthma patients (r = 0.174, P = 0.057; r = -0.138, P = 0.221, respectively). However, a significant negative correlation was found between the salivary free melatonin and cortisol levels in moderate-to-severe persistent asthma patients (r = -0.275, P = 0.013). CONCLUSIONS: There were lower salivary free melatonin and cortisol levels in asthmatic patients. A significant negative correlation was found between melatonin and cortisol levels in moderate-to-severe persistent asthma patients. Furthermore, the reciprocal inhibition of melatonin and cortisol was closely associated with the status of disease in asthmatic patients.

Semiconducting polymer dots with monofunctional groups.

This communication describes an approach for preparing monovalent semiconducting polymer dots (mPdots) with a size of 5 nm where each mPdot was composed of precisely a single active functional group.

Highly luminescent, fluorinated semiconducting polymer dots for cellular imaging and analysis.

A highly fluorescent fluorinated semiconducting polymer dot (Pdot) with a quantum yield of up to 49% was developed. The fluorinated Pdot was eight times brighter in cell-labeling applications than its non-fluorinated counterpart, and was rod shaped rather than spherical.

Importance of having low-density functional groups for generating high-performance semiconducting polymer dots.

Semiconducting polymers with low-density side-chain carboxylic acid groups were synthesized to form stable, functionalized, and highly fluorescent polymer dots (Pdots). The influence of the molar fraction of hydrophilic side-chains on Pdot properties and performance was systematically investigated. Our results show that the density of side-chain carboxylic acid groups significantly affects Pdot stability, internal structure, fluorescence brightness, and nonspecific binding in cellular labeling. Fluorescence spectroscopy, single-particle imaging, and a dye-doping method were employed to investigate the fluorescence brightness and the internal structure of the Pdots. The results of these experiments indicate that semiconducting polymers with low density of side-chain functional groups can form stable, compact, and highly bright Pdots as compared to those with high density of hydrophilic side-chains. The functionalized polymer dots were conjugated to streptavidin (SA) by carbodiimide-catalyzed coupling and the Pdot-SA probes effectively and specifically labeled the cancer cell-surface marker Her2 in human breast cancer cells. The carboxylate-functionalized polymer could also be covalently modified with small functional molecules to generate Pdot probes for click chemistry-based bio-orthogonal labeling. This study presents a promising approach for further developing functional Pdot probes for biological applications.

Stable functionalization of small semiconducting polymer dots via covalent cross-linking and their application for specific cellular imaging.

A facile cross-linking strategy covalently links functional molecules to semiconducting polymer dots (Pdots) while simultaneously providing functional groups for biomolecular conjugation. In addition to greatly enhanced stability, the formed Pdots are small (<10 nm), which can be difficult to achieve with current methods but is highly desirable for most biological applications. These characteristics are significant for improving labeling efficiency and sensitivity in cellular assays that employ Pdots.

Alterations in circadian rhythms of melatonin and cortisol in patients with bronchial asthma.

AIM: To investigate the possible relationships between alterations in circadian rhythm of melatonin, cortisol and bronchial asthma. METHODS: Salivary melatonin and cortisol were measured simultaneously by radioimmunoassay in 10 mild intermittent or persistent patients, 11 moderate-to-severe persistent asthma patients, and 15 control subjects. Twelve salivary samples were collected in a series during a 24-h period in each subject. RESULTS: The results showed overall lower levels of salivary melatonin in asthma patients compared with control subject (P<0.01). The amplitude, peak-level, and baseline of salivary melatonin were significantly lower in mild intermittent or persistent (P<0.01, P<0.05) and moderate-to-severe persistent asthma patients (P<0.01) compared with control group. The 24-h mean level of salivary cortisol was greatly lower and the acrophase was markedly delayed in patients with mild intermittent or persistent asthma (P<0.01) and moderate-to-severe persistent asthma (P< 0.05, P<0.01) compared with control subject. CONCLUSION: Disordered circadian rhythms of salivary melatonin and cortisol were found in asthma patients, which may be involved in the pathogenesis of bronchial asthma.

Alterations in the circadian rhythm of salivary melatonin begin during middle-age.

To investigate whether free melatonin may be better suited to reveal age-related changes, we studied the circadian rhythm alterations in saliva melatonin levels during aging. Special attention was paid to the question as to how the free melatonin rhythms change in aging and when such changes take place. A total of 52 healthy volunteers participated in the study consisting of young, middle-aged, old and the oldest groups. In each subject, a total of 12 time-point salivary melatonin samples was taken over 24 hr. Of the 52 data sets, 51 exhibited significant circadian rhythm over 24 hr by using the base cosine function analysis to fit the data. A clear circadian rhythm of salivary melatonin was present in all age groups. The decline in nocturnal peak levels (amplitude) in salivary melatonin was found in old and the oldest subjects. Both the old and the oldest subjects showed an increased daytime (baseline) melatonin levels. The off-set melatonin levels were more than two times higher in the oldest group than that in the other groups indicating a delayed phase of salivary melatonin. Most strikingly, we found that a step-wise decrease in the circadian rhythms of saliva melatonin occurred early in life, around 40 yr of ages. The middle-aged subjects had only 60% of the amplitude of the young subjects. In addition, the middle-aged subjects showed the longest peak levels duration and the lowest daytime melatonin levels. The present study showed that the alterations in the circadian rhythms of salivary melatonin begin during middle-age. Our results showed that salivary melatonin measurement is a reliable, sensitive and easy method to monitor changes in the circadian rhythms of melatonin during the course of aging.

A pegylated liposomal platform: pharmacokinetics, pharmacodynamics, and toxicity in mice using doxorubicin as a model drug.

Aims were to observe pharmacokinetics, pharmacodynamics, and toxicity for constructing a Sino-pegylated liposomal platform. Human hepatocarcinoma cells (Bel7402) and murine hepatocarcinoma cells (H(22)) were used for the cytotoxicity assay and the in vivo solid xenograft tumor model in mice, respectively. Pharmacokinetic results in mice showed that the pegylated liposomal doxorubicin markedly prolonged the blood circulation of doxorubicin. Elimination half-time (T(1/2,gamma)) of pegylated, regular liposomal doxorubicin and free doxorubicin were 46.09 +/- 14.44, 26.04 +/- 3.34, and 23.72 +/- 5.13 h, respectively. The area under the concentration-time curves (AUC(0- infinity )) (h. microg/g) of the pegylated and regular liposomal doxorubicin were 6.8- and 2.6-fold higher than that of free doxorubicin, respectively. Cytotoxicity and antitumor activity in vivo indicated that activity of the pegylated liposomal doxorubicin was higher than that of the regular or the free one, respectively. After two weeks of tail intravenous injection of the pegylated liposomal doxorubicin at a single dose of 10 mg/kg, no significant damage was observed in gastric, intestinal mucosa, and heart muscle, but pronounced damages were found in the control group after dosing free doxorubicin. The results demonstrate that the pegylated liposomes improve the efficacy of toxics and reduce the toxicity, therefore providing favorable evidence for building a pegylated liposomal platform.