Electrochemiluminescence methods using CdS quantum dots in aptamer-based thrombin biosensors: a comparative study.

The detection of thrombin by using CdS nanocrystals (CdS NCs), gold nanoparticles (AuNPs) and luminol is investigated in this work. Thrombin is detected by three methods. One is called the quenching method. It is based on the quenching effect of AuNPs on the yellow fluorescence of CdS NCs (with excitation/emission wavelengths of 355/550 nm) when placed adjacent to CdS NCs. The second method (called amplification method) is based on an amplification mechanism in which the plasmonics on the AuNPs enhance the emission of CdS NCs through distance related Förster resonance energy transfer (FRET). The third method is ratiometric and based on the emission by two luminophores, viz. CdS NCs and luminol. In this method, by increasing the concentration of thrombin, the intensity of CdS NCs decreases, while that of luminol increases. The results showed that ratiometric method was most sensitive (with an LOD of 500 fg.mL-1), followed by the amplification method (6.5 pg.mL-1) and the quenching method (92 pg.mL-1). Hence, the latter is less useful. Graphical abstract Schematic representation of three different methods (quenching, amplification and ratiometric) were applied for detection of thrombin via aptasensor. The CdS nanocrystals, streptavidin (Str) coated AuNPs and also Str-luminol coated AuNPs were used for the construction steps of the electrochemiluminescence (ECL)-based biosensor.

In situ stress estimation in quantitative micro-elastography.

In quantitative micro-elastography (QME), a pre-characterized compliant layer with a known stress-strain curve is utilized to map stress at the sample surface. However, differences in the boundary conditions of the compliant layer when it is mechanically characterized and when it is used in QME experiments lead to inconsistent stress estimation and consequently, inaccurate elasticity measurements. Here, we propose a novel in situ stress estimation method using an optical coherence tomography (OCT)-based uniaxial compression testing system integrated with the QME experimental setup. By combining OCT-measured axial strain with axial stress determined using a load cell in the QME experiments, we can estimate in situ stress for the compliant layer, more accurately considering its boundary conditions. Our proposed method shows improved accuracy, with an error below 10%, compared to 85% using the existing QME technique with no lubrication. Furthermore, demonstrations on hydrogels and cells indicate the potential of this approach for improving the characterization of the micro-scale mechanical properties of cells and their interactions with the surrounding biomaterial, which has potential for application in cell mechanobiology.

Polarization properties of retinal blood vessel walls measured with polarization sensitive optical coherence tomography.

A new method based on polarization-sensitive optical coherence tomography (PS-OCT) is introduced to determine the polarization properties of human retinal vessel walls, in vivo. Measurements were obtained near the optic nerve head of three healthy human subjects. The double pass phase retardation per unit depth (DPPR/UD), which is proportional to the birefringence, is higher in artery walls, presumably because of the presence of muscle tissue. Measurements in surrounding retinal nerve fiber layer tissue yielded lower DPPR/UD values, suggesting that the retinal vessel wall tissue near the optic nerve is not covered by retinal nerve fiber layer tissue (0.43°/µm vs. 0.77°/µm, respectively). Measurements were obtained from multiple artery-vein pairs, to quantify the different polarization properties. Measurements were taken along a section of the vessel wall, with changes in DPPR/UD up to 15%, while the vessel wall thickness remained relatively constant. A stationary scan pattern was applied to determine the influence of involuntary eye motion on the measurement, which was significant. Measurements were also analyzed by two examiners, with high inter-observer agreement. The measurement repeatability was determined with measurements that were acquired during multiple visits. An improvement in accuracy can be achieved with an ultra-broad-bandwidth PS-OCT system since it will provide more data points in-depth, which reduces the influence of discretization and helps to facilitate better fitting of the birefringence data.

The interaction between the light source dose and caspase-dependent and -independent apoptosis in human SK-MEL-3 skin cancer cells following photodynamic therapy with zinc phthalocyanine: A comparative study.

The aim of this study is to determine the behavior of relative expression of Bcl-2, caspase-8, caspase-9, and caspase-3 genes of/in SK-MEL-3 cancer cells and explore molecular mechanisms responsible for the apoptosis response during an in vitro photodynamic therapy (PDT) with Zinc Phthalocyanine (ZnPc) using different doses of the light source. In this study, firstly the cytotoxic effects of ZnPc-PDT on SK-MEL-3 cells were evaluated. By irradiating the laser, ZnPc induced a significant amount of apoptosis on SK-MEL-3 cells in three IC50s including 0.064±0.01, 0.043±0.01, and 0.036±0.01µg/mL at the doses of 8, 16, and 24J/cm2, respectively. Moreover, flow cytometry and QRT-PCR experiments were done. The high percentage of apoptotic cells was seen in the early apoptosis stage. The expression of Bcl-2 and caspase-8 genes at all doses of laser experienced an obvious reduction in comparison to the control group. On the other hand, although the expression of caspase-9 and caspase-3 genes remains almost constant at 8J/cm2, but they faced an increment at 16 and 24J/cm2 doses. These data reveal caspase-dependent apoptosis in high and caspase-independent apoptosis in low doses of laser. Based on the results of present work, it can be suggested that the dose of the light source is a key factor in induction of caspase-dependent and caspase-independent apoptosis pathways following PDT.

Effects of continuous wave and fractionated diode laser on human fibroblast cancer and dermal normal cells by zinc phthalocyanine in photodynamic therapy: A comparative study.

In this experimental study, cancer and normal cells behavior during an in vitro photodynamic therapy (PDT) under exposure of continuous wave (CW) and fractionated mode of laser with different irradiation power and time intervals was compared and investigated. At the first, human fibroblast cancer cell line (SW 872) and human dermal normal (HFFF2) cell line were incubated with different concentrations of zinc phthalocyanine (ZnPc), as a PDT drug. The cells, then, were irradiated with a 675nm diode laser and the cell viability was evaluated using MTT assay. Under optimized conditions, the viability of the cancer cells was eventually reduced to 3.23% and 13.17%, and that of normal cells was decreased to 20.83% and 36.23% using CW and fractionated diode lasers, respectively. In general, the ratio of ZnPc LD50 values for the normal cells to the cancer cells with CW laser was much higher than that of the fractionated laser. Subsequently, cancer cells in comparison with normal ones were found to be more sensitive toward the photodynamic damage induced by ZnPc. In addition, treatment with CW laser was found to be more effective against the cancer cells with a lower toxicity to the normal cells compared with the fractionated diode laser.

Highly sensitive electrochemiluminescence detection of p53 protein using functionalized Ru-silica nanoporous@gold nanocomposite.

A simple, rapid response time and ultrahigh sensitive electrochemiluminescence (ECL) immunosensor based on Ru(bpy)3(2+)doped silica doped AuNPs (Ru-Si@Au nanocomposite) was developed for detection of p53 protein, a well-known tumor suppressor. The immunosensor was constructed using biotinylated capture antibody, immobilized on the glassy carbon electrode (GCE) using streptavidin modified-gold nanoparticles/thiolated graphene oxide, followed by its conjugation with the Ru-silica@Au nanocomposite labeled secondary antibody to form a sandwich-type immunocomplex. The use of Ru-Si@Au nanocomposites led to a remarkable increase in the ECL intensity and, thus, the sensitivity of the method. Under the optimized conditions, the linear range of the proposed p53 immunosensor was found between 0.2 and 200 pM with a calculated limit of detection of 22.8 fM. The selectivity and reproducibility of the immunosensor was also investigated and the results showed high specificity and great stability in detecting of p53. Moreover, the ECL immunosensor was successfully applied for quantification of p53 protein in the human spiked serum samples and more importantly in the human normal and cancer skin fibroblast cells showing much satisfactory result compared with the ELISA method. The proposed immunosensor reported herein offers a considerable potential in early detection of cancer and clinical diagnosis and provides a new platform for biomarker detection.