Siu Kai Kong

Highly sensitive differential phase-sensitive surface plasmon resonance biosensor based on the Mach–Zehnder configuration

A high-sensitivity surface plasmon resonance (SPR) biosensor based on the Mach-Zehnder interferometer design is presented. The novel feature of the new design is the use of a Wollaston prism through which the phase quantities of the p and s polarizations are interrogated simultaneously. Since SPR affects only the p polarization, the signal due to the s polarization can be used as the reference. Consequently, the differential phase between the two polarizations allows us to eliminate all common-path phase noise while keeping the phase change caused by the SPR effect. Experimental results obtained from glycerin-water mixtures indicate that the sensitivity limit of our scheme is 5.5 x 10(-8) refractive-index units per 0.01 degrees phase change. To our knowledge, this is a significant improvement over previously obtained results when gold was used as the sensor surface. Such an improvement in the sensitivity limit should allow SPR biosensors to become a possible replacement for conventional biosensing techniques based on fluorescence. Monitoring of the bovine serum albumin (BSA) binding reaction with BSA antibodies is also demonstrated.

Effects of polyphyllin D, a steroidal saponin in Paris Polyphylla, in growth inhibition of human breast cancer cells and in xenograft

Paris Polyphylla is a traditional Chinese Medical herb that has been used in treating cancer for thousands of year. Without studies on the anticancer effects of Paris Polyphylla being initiated before, we have first studied the component of Paris Polyphylla and have spotted out a steroidal saponin, polyphyllin D. As long as the chemical structure and the improved synthesis of polyphyllin D were ascertained, both in vitro to in vivo studies were performed. It was found that treatment of MCF-7 and MDA-MB-231 cells with polyphyllin D resulted in the inhibition of viability and induction of apoptosis in a dose-dependent manner, with an IC50 of 5?M and 2.5?M, respectively, after 48 hours of incubations. Apoptosis of MCF-7 and MDA-MB-231 cells by polyphyllin D was evidenced by the occurrence of DNA fragmentation, formation of a hypodiploid peak in the cell cycle analysis, phosphatidyl-serine externalization and a late loss of membrane integrity. Mechanistically, polyphyllin D dissipates the mitochondrial membrane potential, induces a down-regulation of anti-apoptotic Bcl-2 expression and an up-regulation of pro-apoptotic Bax expression, and activates caspase-9. These results suggest that polyphyllin D elicits apoptosis through mitochondria dysfunction. In vivo study demonstrated that daily administration of polyphyllin D (2.73 mg/kg body weight) through intravenous injection for ten days in nude mice bearing MCF-7 cells effectively reduced tumor growth for 50% in terms of tumor weight and size, given no significant toxicity in heart and liver to the host. All these findings provide novel insights that polyphyllin D could serve as a candidate in breast cancer treatment.

Polyphyllin D induces apoptosis in human erythrocytes through Ca2+ rise and membrane permeabilization

Polyphyllin D (PD) is a potent anticancer agent isolated from a traditional medicinal herb Paris polyphylla that has been used in China for many years to treat cancer. PD is not a substrate of p-glycoprotein, and it can bypass the multi-drug resistance in cancer cell line R-HepG2. However, the effect of PD on the induction of cell death in human erythrocytes remains unknown. Given that PD is a small molecule that can depolarize the mitochondrial membrane potential and release apoptosis-inducing factor (AIF) in isolated mitochondria, we hypothesized that the apoptogenic effect of PD in human erythrocytes devoid of mitochondria would be minimal. This study therefore tried to evaluate the in vitro effect of PD on hemolysis and apoptosis in human erythrocytes. Apoptosis in human red blood cells (RBCs), also known as eryptosis or erythroptosis, after PD treatment was determined by flow cytometry and confocal microscopy for the phosphatidyl-serine externalization and other apoptosis feature events. False to our prediction, PD caused hemolysis and eryptosis/erythroptosis in human RBCs. Mechanistically, elevation in the cytosolic Ca2+ ion level seems to be a key but not the only mediator in the PD-mediated eryptosis/erythroptosis because depletion of the external Ca2+ could not eliminate the PD effect. Also, PD was able to permeabilize the membrane of RBC ghosts in a way similar to digitonin. Taken together, we report here for the first time the toxicity of PD in human RBCs as well as its underlying mechanism for the hemolysis and eryptosis/erythroptosis.

Detecting Phase Shifts in Surface Plasmon Resonance: A Review

Under certain conditions, a surface plasmon wave along a metal-dielectric interface can be excited by an optical beam. The reflected optical beam will then undergo changes in both intensity and phase. As the level of intensity or phase change is quite sensitive to the coupling conditions such as the molecule concentration on the metal surface, this phenomenon has been utilized for label-free detection of biological species and characterization of molecular interactions during the last two decades. Currently, most of the commercial surface plasmon resonance (SPR) sensors rely on the detection of absorption dip in angular or wavelength spectrum. However, recent researches have shown that phase detection has the potential to achieve lower limit of detection (LoD) and higher throughput. This paper, thus, intends to review various schemes and configurations for SPR phase detection. The performance advantages and disadvantages of various schemes will be emphasized. It is hoped that this paper will provide some insights to researchers interested in SPR sensing and help them to develop SPR sensors with better sensitivity and higher throughput.

Pheophorbide a, an active compound isolated from Scutellaria barbata, possesses photodynamic activities by inducing apoptosis in human hepatocellular carcinoma

Photodynamic therapy (PDT) is an effective treatment for cancer by inducing apoptosis or necrosis in the target cells. Pheophorbide a (Pa), a chlorophyll derivative, is a photosensitzier which can induce significant anti-proliferative effects in a number of human cancer cell lines. This study investigated the action mechanism of Pa-mediated photodynamic therapy (Pa-PDT) on the human hepatocellular carcinoma, Hep3B cells. Pa-PDT significantly inhibited the growth of Hep3B cells with an IC50 value of 1.5?M. Intracellular ROS level was increased in Pa-PDT treated cells and the cytotoxic effect could be reversed when ascorbic acid was applied. Pa was found to be localized in the mitochondria and then induced the target cells to undergo apoptosis, which was confirmed by propidium iodide staining and DNA fragmentation assay. Pa-PDT treatment also led to the depolarization of mitochondrial membrane potential (??m) and a release of cytochrome c from mitochondria to the cytosol. The caspase cascade was activated as shown by a significant decrease of procaspase-3 and –9 in Pa-PDT treated cells in a dose-dependent manner. Furthermore, in nude mice model, Pa-PDT treatment could reduce the tumor size by 57% after 14 days treatment.

Heat shock protein 70 inhibits the nuclear import of apoptosis-inducing factor to avoid DNA fragmentation in TF-1 cells during erythropoiesis

Loss of mitochondrial membrane potential (ΔΨm) and release of AIF (apoptosis‐inducing factor) from mitochondria are key steps in apoptosis. In TF‐1 model, ΔΨm was depolarized with AIF release during erythroid development. Yet, no DNA fragmentation was observed. When ΔΨm depolarization had been blocked, erythropoiesis was suppressed. Interestingly, heat shock protein 70 (Hsp70) was found transiently upregulated during depolarization and it retained AIF in the cytosol to avoid DNA damages. When Hsp inhibitor was added, DNA fragmentation occurred. We show this mechanism for the first time in erythropoiesis how cells with ΔΨm depolarization and AIF release escape apoptosis.

Real-time protein biosensor arrays based on surface plasmon resonance differential phase imaging

This paper reports the application of differential phase surface plasmon resonance (SPR) imaging in two-dimensional (2D) protein biosensor arrays. Our phase imaging approach offers a distinct advantage over the conventional angular SPR technique in terms of utilization efficiency of optical sensor elements in the imaging device. In the angular approach, each biosensor site in the biosensor array requires a linear array of optical detector elements to locate the SPR angular dip. The maximum biosensor density that a two-dimensional imaging device can offer is a one-dimensional SPR biosensor array. On the other hand, the phase-sensitive SPR approach captures data in the time domain instead of the spatial domain. It is possible that each pixel in the captured interferogram represents one sensor site, thus offering high-density two-dimensional biosensor arrays. In addition, our differential phase approach improves detection resolution through removing common-mode disturbances. Experimental results demonstrate a system resolution of 8.8 x 10(-7)RIU (refractive index unit). Real-time monitoring of bovine serum albumin (BSA)/anti-BSA binding interactions at various concentration levels was achieved using a biosensor array. The detection limit was 0.77 microg/ml. The reported two-dimensional SPR biosensor array offers a real-time and non-labeling detection tool for high-throughput protein array analysis. It may find promising applications in protein therapeutics, drug screening and clinical diagnostics.

Concanavalin A induced apoptosis in murine macrophage PU5-1.8 cells through clustering of mitochondria and release of cytochrome c.

Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macrophage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypo-diploid DNA in a dose dependent manner as revealed by flow cytometry; (2) ConA elicited DNA fragmentation and the cytotoxicity of ConA was suppressed by α-D-methylmannoside which blocks the lectin site of ConA; (3) ConA was able to release cytochrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochondria were incubated with ConA, release of cyto c was observed too. Interestingly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC and subsequently with mitotracker red (a probe for mitochondria), co-localization of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and released cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. On the other hand, introduction of specific antibody against cyto c into PU5-1.8 cells suppressed the ConA-mediated cell death. Taken together, our results indicate that ConA induced apoptosis in PU5-1.8 cells through mitochondrial clustering and release of cyto c and the release of cyto c was sufficient to elicit apoptosis in PU5-1.8 cells.

Photodynamic therapy inhibits p-glycoprotein mediated multidrug resistance via JNK activation in human hepatocellular carcinoma using the photosensitizer pheophorbide a

BackgroundMultidrug resistance (MDR) is frequently observed after prolonged treatment in human hepatoma with conventional anti-tumor drugs, and photodynamic therapy (PDT) is a recently suggested alternative to overcome MDR. The therapeutic potential of PDT was evaluated in a multidrug resistance (MDR) human hepatoma cell line R-HepG2 with photosensitizer pheophorbide a (Pa).ResultsOur results demonstrated that intracellular accumulation of Pa was not reduced by the overexpression of P-glycoprotein. Pa-based PDT (Pa-PDT) significantly inhibited the growth of R-HepG2 cells with an IC50 value of 0.6 μM. Mechanistic study demonstrated that genomic DNA fragmentation and phosphatidylserine externalization occurred where increase of intracellular singlet oxygen level triggers the phosphorylation of c-Jun N-terminal Kinase (JNK) and leads to activation of intrinsic apoptotic caspases cascade during the Pa-PDT treatment. The cytotoxicity of Pa-PDT, accumulation of sub-G1 population, and depolarization of mitochondrial membrane could be inhibited by JNK inhibitor in the Pa-PDT treated cells. Interestingly, the Pa-PDT induced JNK activation showed inhibitory effect on MDR by the down-regulation of P-glycoprotein in R-HepG2 cells in a dose-dependent manner. In addition, significant reduction of tumor size was obtained in Pa-PDT treated R-HepG2-bearing nude mice with no significant damages in liver and heart.ConclusionIn summary, our findings provided the first evidence that PDT could inhibit the MDR activity by down-regulating the expression of P-glycoprotein via JNK activation using pheophorbide a as the photosensitizer, and our work proved that Pa-PDT inhibited the growth of MDR hepatoma cells by mitochondrial-mediated apoptosis induction.

Surface-enhanced Raman scattering biosensor for DNA detection on nanoparticle island substrates

We present a study on the surface-enhanced Raman scattering (SERS) properties of Ag nanoparticle island substrates (NIS) and their applications for target oligonucleotide (OND) detection. It has been found that the surface nanostructure of NIS samples can be controlled with a good degree of reproducibility, and a high SERS enhancement can be achieved when the peak extinction wavelength of NIS is tuned to a spectral window (approximately 60 nm) between the excitation wavelength and the scattered Raman wavelength. The highest SERS enhancement was obtained from the NIS substrates with a nominal thickness of 50 A. Detection of target OND was performed with a sandwich format in which the target OND was hybridized both to a capture OND immobilized on the NIS substrate, and a detection OND conjugated with a Raman-active dye for SERS signal generation. We compare the detection performance of two strategies based on the use of the detection OND with or without the gold nanoparticle (Au-NP). Our results confirm that, when the detection OND is coupled to the Au-NP, a better sensitivity for the target OND detection, in terms of a wider dynamic range and a lower detection limit (0.4 fM versus 1 nM without Au-NP), would be achieved.