Light Activation Methods Research Articles

A one-pot method based on rolling circle amplification and light‐activated CRISPR/Cas12a reaction for simple and highly sensitive detection of Staphylococcus aureus

Developing robust methods that achieve simple and highly sensitive detection of Staphylococcus aureus (S. aureus) is still a challenge. Isothermal nucleic acid amplification reactions-triggered CRISPR/Cas12a strategies offer promising sensing tools for bacteria detection, but have several issues that need to be solved, such as incompatible reaction systems, compromised sensitivity, and multiple reaction steps. Here, we develop a light-activated one-pot method based on rolling circle amplification-triggered CRISPR/Cas12a (RCA-Cas12a) for analyzing S. aureus directly. In the one-pot assay, the Cas12a reaction is started under light irradiation only after the RCA reaction is completed, thus avoiding interference between the two systems. The light-activated method is simple to operate, and its sensitivity is two orders of magnitude higher than that of the traditional one-pot assay. Accurately identifying contaminated samples from 120 real samples (serum, milk, and broth) prepared in a double-blind way demonstrates the good application capacity of the light-activated one-pot method. The method has a shorter analysis time, fewer operation steps, and higher accuracy in identifying contaminated samples compared with bacterial culture. Thus, the light-activated one-pot RCA-Cas12a method represents a simple tool for sensitive and specific detection of S. aureus in food and clinical samples.

Coupling Persulfate-Based AOPs: A Novel Approach for Piroxicam Degradation in Aqueous Matrices

The activated persulfate degradation of piroxicam, a non-steroidal anti-inflammatory drug (NSAID) belonging to oxicams, was investigated. Persulfate was activated with thermal energy or (UV-A and simulated solar) irradiation. Using 250 mg/L sodium persulfate at 40 °C degraded almost completely 0.5 mg/L of piroxicam in 30 min. Increasing piroxicam concentration from 0.5 to 4.5 mg/L decreased its removal. The observed kinetic constant was increased almost ten times from 0.077 to 0.755 min−1, when the temperature was increased from 40 to 60 °C, respectively. Process efficiency was enhanced at pH 5–7. At ambient conditions and 30 min of irradiation, 94.1% and 89.8% of 0.5 mg/L piroxicam was removed using UV-A LED or simulated solar radiation, respectively. Interestingly, the use of simulated sunlight was advantageous over UV-A light for both secondary effluent, and 20 mg/L of humic acid solution. Unlike other advanced oxidation processes, the presence of bicarbonate or chloride in the range 50–250 mg/L enhanced the degradation rate, while the presence of humic acid delayed the removal of piroxicam. The use of 0.5 and 10 g/L of methanol or tert-butanol as radical scavengers inhibited the reaction. The coupling of thermal and light activation methods in different aqueous matrices showed a high level of synergy. The synergy factor was calculated as 68.4% and 58.4% for thermal activation (40 °C) coupled with either solar light in 20 mg/L of humic acid or UV-A LED light in secondary effluent, respectively.

Light Activation and Thermocycling Methods on the Shear Bond Strength of Brackets Bonded to Porcelain Surfaces.

The present study was evaluated the effect of different light activation and thermocycling methods on the shear bond strength (SBS) and on the adhesive remnant index (ARI) of metal brackets bonded to feldspathic ceramic. Hundred metal brackets were bonded to 20 porcelain cylinders, divided into four groups (n=25) based on light activation and thermocycling processes. The cylinders were etched with 10% hydrofluoric acid for 60 s and coated with two layers of silane. The brackets were bonded with Transbond XT composite resin. Light activation in Groups 1 and 3 was performed during 3 s using the VALO Ortho Cordless appliance with irradiance 3,200 mW/cm2 and in Groups 2 and 4 for 40 s using Optilight Max appliance with irradiance 1,200 mW/cm². The samples were stored in deionized water at 37°C for 24 h and the samples from Groups 1 and 2 were submitted to the SBS test at a rate of 1 mm/min, whereas the samples from Groups 3 and 4 were submitted to 7,000 thermal cycles (5°/55°C) before to the SBS test. The data were assessed by two-way analysis of variance and by Tukey's test (a=0.05). No significant difference was observed between SBS means in the different light activation devices used. The samples subjected to thermocycling revealed lower SBS values (p≤0.05). There was predominance of score 0 for ARI in all groups. Therefore, the different light activation methods did not interfere in SBS, but thermocycling reduced SBS.

Sealing of Corneal Lacerations Using Photoactivated Rose Bengal Dye and Amniotic Membrane.

Watertight closure of perforating corneoscleral lacerations is necessary to prevent epithelial ingrowth, infection, and potential loss of the eye. Complex lacerations can be difficult to treat, and repair with sutures alone is often inadequate. In this study, we evaluated a potentially sutureless technology for sealing complex corneal and scleral lacerations that bonds the amniotic membrane (AM) to the wound using only green light and rose bengal dye. The AM was impregnated with rose bengal and then sealed over lacerations using green light to bond the AM to the deepithelialized corneal surface. This process was compared with suture repair of 3 laceration configurations in New Zealand White rabbits in 3 arms of the study. A fourth study arm assessed the side effect profile including viability of cells in the iris, damage to the blood-retinal barrier, retinal photoreceptors, retinal pigment epithelium, and choriocapillaris in Dutch Belted rabbits. Analyses of the first 3 arms revealed a clinically insignificant increase in polymorphonuclear inflammation. In the fourth arm, iris cells appeared unaffected and no evidence of breakdown of the blood-retinal barrier was detected. The retina from green light laser-treated eyes showed normal retinal pigment epithelium, intact outer segments, and normal outer nuclear layer thickness. The results of these studies established that a light-activated method to cross-link AM to the cornea can be used for sealing complex penetrating wounds in the cornea and sclera with minimal inflammation or secondary effects.

Effect of Adhesive Restoration and Bleaching Technique on the Concentration of Hydrogen Peroxide In the Pulp Chamber.

This study aimed to quantify the concentration of hydrogen peroxide into the pulp chamber in the presence or absence of adhesive enamel restorations and to analyze the resin-dentin interface of bleached groups. Bovine incisors (120) were randomly divided into three groups according to enamel treatment (n=40 each): (1) enamel without restoration (control); (2) enamel cavities (3 mm diameter × 1.5 mm depth) restored with a silorane-based (SB) system; or (3) enamel cavities (3 mm diameter × 1.5 mm depth) restored with a dimethacrylate-based (DB) system. Restorations were thermocycled, and all groups were submitted to one application of 35% hydrogen peroxide (HP) agent for 45 minutes and subjected to four light activation methods (n=10 each): without light, light-emitting diode (LED), LED/diode laser, or halogen light. Acetate buffer solution was placed into the pulp chamber before bleaching, and this solution was collected to spectrophotometrically determine the concentration of HP that reached the pulp chamber after bleaching. Rhodamine B was added to the HP agent and applied on additional enamel samples of each group for 24 hours. Samples were sectioned mesiodistally, and the bleaching agent was traced using confocal microscopy. According to two-way analysis of variance and Tukey test (α=0.05), the HP concentration in the pulp chamber of the control group was significantly lower than that of the SB group (p<0.05), regardless of light activation. No differences were observed between DB and SB groups and between control and DB groups, except for the DB halogen light activated group, which exhibited higher HP intrapulpal concentration (p<0.05). Confocal microscopy exhibited HP diffusion through the interface of the SB and DB restored groups as well as enamel prisms in the control group. The SB restorative system increased the HP diffusion into the pulp chamber, but HP was able to diffuse even in the absence of enamel restorations.

광활성 유무가 치아미백과 치아표면 및 치수 내 온도에 미치는 영향

Received : 17 April, 2014 Revised : 21 July, 2014 Accepted : 11 August, 2014 ABSTRACT Objectives : The purpose of this study was to investigate of the color change, tooth surface and intra-pulpal temperature of tooth bleaching by light activation Methods : Forty-eight extracted bovine teeth were immersed into a tea solution for 24 hours. The specimens were randomly divided into four groups(n=15):(G1) 15% HP + without light activation, (G2) 15% HP + light activation, (G3) 25% HP + without light activation, (G4) 25% HP + light activation. All specimens were bleached for 15 minutes three times. The spectrophotometer (CM-2600d, Konica Minolta, Osaka, Japan) was used including before bleaching, immediately after bleaching, 1 week, 1 and 3 months after the end of bleaching. The temperature rise were measured in the pulpal chamber and tooth surface with a digital thermocouple thermometer(Termopar Digital Multimeter, Tektronix DMM916, USA). Between the tested time points, the specimens were stored in distilled water. The data were analyzed by ANOVA, t-test and Tukey’s post hoc test set at 0.05. Corresponding Author Youn-Soo Shim Department of Dental Hygiene Sunmoon University 70 Sunmoon-ro, Tangjeong-myeon Asan-si, Chungnam 336-708, Korea. Tel : +82-41-530-2740 +82-10-3404-2171 Fax : +82-41-530-2726 E-mail : shim-21@hanmail.net Results : There was no significant color change by the use of light after the bleaching treatment(p>0.05). The dental bleaching treatments of teeth with 15% HP and 25% HP did not seem to be more effective when light source was used. There was no difference in color stability between groups within three month(p>0.05). There was an increase in tooth surface and pulp temperature, but it was not sufficient to cause damage to the pulp. Conclusions :The use of light activation has no obvious effective impact on the tooth bleaching effect.

Small Molecule-Initiated Light-Activated Semiconducting Polymer Dots: An Integrated Nanoplatform for Targeted Photodynamic Therapy and Imaging of Cancer Cells

Photodynamic therapy (PDT) is a noninvasive and light-activated method for cancer treatment. Two of the vital parameters that govern the efficiency of PDT are the light irradiation to the photosensitizer and visual detection of the selective accumulation of the photosensitizer in malignant cells. Herein, we prepared an integrated nanoplatform for targeted PDT and imaging of cancer cells using folic acid and horseradish peroxidase (HRP)-bifunctionalized semiconducting polymer dots (FH-Pdots). In the FH-Pdots, meta-tetra(hydroxyphenyl)-chlorin (m-THPC) was used as photosensitizer to produce cytotoxic reactive oxygen species (ROS); fluorescent semiconducting polymer poly[2-methoxy-5-((2-ethylhexyl)oxy)-p-phenylenevinylene] was used as light antenna and hydrophobic matrix for incorporating m-THPC, and amphiphilic Janus dendrimer was used as a surface functionalization agent to conjugate HRP and aminated folic acid onto the surface of FH-Pdots. Results indicated that the doped m-THPC can be simultaneously excited by the on-site luminol-H2O2-HRP chemiluminescence system through two paths. One is directly through chemiluminescence resonance energy transfer (CRET), and the other is through CRET and subsequent fluorescence resonance energy transfer. In vitro PDT and specificity studies of FH-Pdots using a standard transcriptional and translational assay against MCF-7 breast cancer cells, C6 glioma cells, and NIH 3T3 fibroblast cells demonstrated that cell viability decreased with increasing concentration of FH-Pdots. At the same concentration of FH-Pdots, the decrease in cell viability was positively relevant with increasing folate receptor expression. Results from in vitro fluorescence imaging exhibited that more FH-Pdots were internalized by cancerous MCF-7 and C6 cells than by noncancerous NIH 3T3 cells. All the results demonstrate that the designed semiconducting FH-Pdots can be used as an integrated nanoplatform for targeted PDT and on-site imaging of cancer cells.

Visible light driven photocatalysis mediated via ligand-to-metal charge transfer (LMCT): an alternative approach to solar activation of titania

Visible light harvesting or utilization through semiconductor photocatalysis is a key technology for solar chemical conversion processes. Although titania nanoparticles are popular as a base material of photocatalysis, the lack of visible light activity needs to be overcome. This mini-review is focused on an uncommon approach to visible light activation of titania: the ligand-to-metal charge transfer (LMCT) that takes place between TiO2 nanoparticles and surface adsorbates under visible light irradiation. We discuss a basic concept of photoinduced LMCT and the recent advances in LMCT-mediated visible light photocatalysis which has been applied in environmental remediation and solar energy conversion. Although the LMCT processes have been less investigated and limited in photocatalytic applications compared with other popular visible light activation methods such as impurity doping and dye sensitization, they provide lots of possibilities and flexibility in that a wide variety of organic or inorganic compounds can form surface complexes with TiO2 and introduce a new absorption band in the visible light region. The LMCT complexes may serve as a visible light sensitizer that initiates the photocatalytic conversion of various substrates or the self-degradation of the ligand complexes (usually pollutants) themselves. We summarized and discussed various LMCT photocatalytic systems and their characteristics. The LMCT-mediated activation of titania and other wide bandgap semiconductors has great potential to be developed as a more general method of solar energy utilization in photocatalytic systems. More systematic design and utilization of LMCT complexes on semiconductors are warranted to advance the solar-driven chemical conversion processes.

Photochemical tissue bonding: A potential strategy for treating limbal stem cell deficiency

To determine the feasibility of attaching human amniotic membrane (HAM), pre-cultured with limbal stem cells (LSCs), to cornea using a novel, light-activated tissue bonding method. LSCs were isolated from rabbit eyes, and then cultured on de-epithelialized HAM to create grafts (HAM/LSC). These were then transplanted onto rabbit eyes with surgically created limbal stem cell deficiency (LSCD). The grafts were secured either by sutures or by a light-activated method called photochemical tissue bonding (PTB). Outcomes included corneal opacity, inflammation, neovascularization, and collagen alignment. The isolated and cultured cells were verified to be LSCs based on their K19+/intergrin β1+/P63+/K3 profile. Securing the HAM/LSC graft with PTB provided better outcomes. At 28 days post-surgery, the corneal opacity scores were significantly lower after securing the graft with PTB compared with suture attachment (0.8 ± 0.5 vs. 1.8 ± 0.5, P < 0.01). Similarly, neovascularization scores were lower after PTB (0.8 ± 0.5 vs. 1.5 ± 0.6, P < 0.01). Quantification of MPO and CD31 levels from immunofluorecent staining indicated that PTB stimulated less neutrophil infiltration (5.3 ± 2.2 vs. 13.3 ± 3.1, P < 0.01) and less new blood vessels formation (2.0 ± 0.8 vs. 6.3 ± 1.3, P < 0.01) at the wound site. The collagen alignment in PTB-treated corneas, as shown by immunofluorescence and second harmonic generation image, was better organized in the PTB-treated group than in the suture group. Bonding LSC grafts with PTB produced improved outcomes compared to suture attachment. This light-activated method is a promising modality for treating patients with LSCD.

Phototoxicity is not associated with photochemical tissue bonding of skin

We have developed a light-activated method called photochemical tissue bonding (PTB) for closing wounds using green light and a photosensitizing dye (Rose Bengal-RB) to initiate photochemical crosslinking of wound surface proteins. These studies were designed to determine whether RB causes phototoxicity during closure of skin incisions with PTB. RB phototoxicity was evaluated after sealing incisions in porcine skin ex vivo and rabbit skin in vivo using PTB (1 mM RB, 100 J/cm(2), 532 nm, 0.3 or 0.5 W/cm(2).) Dead cells were identified by pyknotic nuclei and eosinophilic cytoplasm on H&E-stained sections. The influence on RB phototoxicity of penetration of RB into the wound wall (by confocal microscopy), RB concentration in the tissue (by extraction), and fluence of 532 nm reaching depths in skin (calculated from skin optical properties) were investigated. No significant differences were found in the percent dead cells in PTB-treated and control incisions in porcine skin at 24 hours or in rabbit skin at 2 hours and 3 and 7 days after surgery. RB was retained in a approximately 100 microm wide band next to the wound wall. The mean RB concentration within this band was 0.42+/-0.03 mM. Monte Carlo modeling of light distribution indicated that the fluence rate decreased from the subsurface peak to 0.5 W/cm(2) in the mid-dermis (approximately 350 microm.) In vitro RB phototoxicity to dermal fibroblasts yielded an LD(50) of 0.50+/-0.09 J/cm(2) when the cells contained 0.46 mM RB. PTB does not cause phototoxicity when used to repair skin wounds even though the RB concentration and 532 nm fluence in the mid-dermis during PTB are much greater than the LD(50) for RB phototoxicity in vitro. These results indicate that phototoxicity is not a concern when using PTB for tissue repair.

Calculation of contraction stresses in dental composites by analysis of crack propagation in the matrix surrounding a cavity

Objectives Polymerization contraction of dental composite produces a stress field in the bonded surrounding substrate that may be capable of propagating cracks from pre-existing flaws. The objectives of this study were to assess the extent of crack propagation from flaws in the surrounding ceramic substrate caused by composite contraction stresses, and to propose a method to calculate the contraction stress in the ceramic using indentation fracture. Methods Initial cracks were introduced with a Vickers indenter near a cylindrical hole drilled into a glass-ceramic simulating enamel. Lengths of the radial indentation cracks were measured. Three composites having different contraction stresses were cured within the hole using one- or two-step light-activation methods and the crack lengths were measured. The contraction stress in the ceramic was calculated from the crack length and the fracture toughness of the glass-ceramic. Interfacial gaps between the composite and the ceramic were expressed as the ratio of the gap length to the hole perimeter, as well as the maximum gap width. Results All groups revealed crack propagation and the formation of contraction gaps. The calculated contraction stresses ranged from 4.2 MPa to 7.0 MPa. There was no correlation between the stress values and the contraction gaps. Significance This method for calculating the stresses produced by composites is a relatively simple technique requiring a conventional hardness tester. The method can investigate two clinical phenomena that may occur during the placement of composite restorations, i.e. simulated enamel cracking near the margins and the formation of contraction gaps.

Self-Assembled Monolayers Directly Attached to Silicon Substrates Formed from 1-Hexadecene by Thermal, Ultraviolet, and Visible Light Activation Methods

Using 1-hexadecene as a precursor, self-assembled monolayers (SAMs) were fabricated on hydrogen-terminated silicon (111) [H-Si(111)] surfaces without forming an interfacial oxide layer on the basis of thermal (180 °C, 2 h), ultraviolet (UV; 500 mW cm-2, 10 h), and visible-light activation (330 mW cm-2, 16 h) processes. As characterized by water contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and ellipsometry, the hexadecyl SAM fabricated by the visible-light process had a highly-ordered molecular arrangement and a closely-packed methyl-terminated surface similar to the SAMs prepared by the thermal and UV activation processes. The photo-irradiation wavelength dependence of the visible-light activation process was further studied at irradiation wavelengths of 400, 550, and 700 nm. The SAM formation reaction was certainly promoted at all the wavelengths, even at 700 nm. However, oxidation of the Si surface became apparent due to the slow rate in SAM growth, and thus the monolayer coverage of SAM at 700 nm became smaller. The reaction rate became faster with the decreasing wavelength for activation, probably due to the increase in the light adsorption coefficient of Si. The excitation of Si, namely, the generation of hole/electron pairs at the Si substrate, is assumed to be the rate-controlling step of the visible-light activation process.

Structural Analysis of Leukotriene C 4 Isomers Using Collisional Activation and 157 nm Photodissociation

The fragmentation of 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid [leukotriene C4 or LTC4 (5, 6)] and its isomeric counterpart LTC4 (14, 15) were studied by low and high-energy collisional induced dissociation (CID) and 157 nm photofragmentation. For singly charged protonated LTC4 precursors, photodissociation significantly enhances the signal intensities of informative fragment ions that are very important to distinguish the two LTC4 isomers and generates a few additional fragment ions that are not usually observed in CID experiments. The ion trap enables MSn experiments on the fragment ions generated by photodissociation. Photofragmentation is found to be suitable for the structural identification and isomeric differentiation of cysteinyl leukotrienes and is more informative than low or high-energy CID. We describe for the first time the structural characterization of the LTC4 (14, 15) isomer by mass spectrometry using CID and 157 nm light activation methods.

Effect of light-activation methods and water storage on the flexural strength of two composite resins and a compomer

The present study evaluated the flexural strength of three composite resins recommended for direct esthetic restorations: a polyacid modified composite (Dyract AP), a unimodal composite resin (Filtek Z250) and a hybrid composite resin (Point 4). The variation factors, apart from the type of composite resin, were the light activation method and the water storage period. The composite resins were light-cured in continuous mode (40 s, 500 mW/cm2) or in ramp mode (0-800 mW/cm2 for 10 s followed by 30 s at 800 mW/cm2) and stored for 24 hours or 30 days in distilled water at 37 degrees C. The data were analyzed by ANOVA and Tukey test for multiple comparisons (alpha = 0.05). The composite resin Z250 presented the highest mean flexural strength (166.74 MPa) and Dyract AP presented the lowest one (129.76 MPa). The storage for 30 days decreased the flexural strength in ramp mode (24 h: 156.64 MPa; 30 days: 135.58 MPa). The light activation method alone did not lead to different flexural strength values.