Sensitive detection of heavy metal stress in aquatic plants by dissolved oxygen-quenched fluorescence/materials movement-induced beam deflection method.

Sensitive detection of heavy metal (HM) stress in aquatic plants by dissolved oxygen (DO)-quenched fluorescence/materials movement-induced beam deflection method is demonstrated. Egeria densa Planchon and Cu2+ were used as a model aquatic plant and HM ion, respectively. Reproducibility and experimental errors of the method were first investigated in a control culture solution only containing 10-6 M Ru (II) complex (Tris (2,2'-bipyridyl) ruthenium (II) chloride) without addition of any fertilizer and Cu2+. Changes of DO concentration (∆DO) and deflection (∆DE) during the monitoring periods were used as parameters to quantitatively evaluate the experimental errors and detection limits. Averages or means ([Formula: see text], [Formula: see text]) and standard deviations (σ∆DO, σ∆DE) of ∆DO and ∆DE in seven control experiments with different aquatic plants sheets during both the respiration and photosynthesis processes were obtained. Next, DO and deflection at the middle vicinities of the aquatic plant were monitored during 2 h of both respiration and photosynthesis in presence of 10-10 ~ 10-6 M Cu2+. Experimental results showed that the aquatic plant began to suffer from the HM stress in some extent in presence of 10-9 M Cu2+. When the concentration of Cu2+ was higher than 10-8 M, changing trends of both DO and deflection with time were not reversed during the respiration and photosynthesis, implying that the materials movements in the physiological activities had been altered greatly. It is demonstrated that the method could sensitively detect the HM stress in the aquatic plants given by nM HM ions in culture solution without addition of a fertilizer. Furthermore, detection limits of the method were quantitatively discussed by considering [Formula: see text] σ∆DO and [Formula: see text] σ∆DE as the minimum detectable changes of DO and deflection caused by the HM stress, respectively.

Improvements on the Fluorescence Quenching/Deflection Method for Real-time in situ Simultaneous Monitoring of Dissolved Oxygen and Material Movement-induced Beam Deflection in the Vicinity of an Aquatic Plant.

Although the newly developed beam deflection/fluorescence detection system for real-time in situ simultaneous monitoring of dissolved oxygen (DO) and material movements in the vicinity of aquatic plants was not only much more sensitive but also could be carried out much more closely to real time than conventional analytical methods that monitor the concentration changes at a bulk solution, it could not be applied to the photosynthesis process of aquatic plants. Here, improvements are reported to enable application of the system to the photosynthesis process. A white-light LED, which was used as a light source for photosynthesis in our previous paper, was replaced by a red-blue LED with wavelength of about 660 and 450 nm. Also, an interference filter of 589 ± 25 nm was placed in front of a photomultiplier tube (PMT). Furthermore, the LED and its electric power supply were placed outside of the dark room for preventing great temperature increases in the photosynthetic experiments. Experimental results showed the DO-quenched fluorescence could be sensitively monitored in both the respiration and photosynthesis processes, while only in the respiration process before the improvements. It is successfully demonstrated that the DO change and material movement-induced beam deflection in the vicinity of the plants in both the respiration and photosynthesis processes could be real-time in situ monitored with high sensitivity.

Real-time in-situ Simultaneous Monitoring of Dissolved Oxygen and Materials Movements at a Vicinity of Micrometers from an Aquatic Plant by Combining Deflection of a Probe Beam and Fluorescence Quenching.

It is desirable to be able to monitor the intake or release of the components at different organs of aquatic plants in real time and in-situ. Here, we report a novel optical detection system that allows for real-time in-situ simultaneous monitoring of the dissolved oxygen and material movements at a vicinity of micrometers from the aquatic plant surface. A blue semiconductor diode-laser was used as the light source of both the probe beam and excitation light for fluorescence. The laser light reflected by a dichroic mirror was focused to a vicinity of the plant/water interface in a culture dish by an objective lens. The distance between the focused laser beam and the plant surface was adjusted by an X-Y-Z micro-stage. Deflection of the probe beam was detected by a position sensor, and fluorescence from the vicinity was monitored by a PMT. A commercial fluorescent DO sensor, which simultaneously monitored temperature, was immersed into the culture dish at about 1 cm away from the aquatic plants. A white-light LED was used to illuminate the aquatic plants in the dish in photosynthesis process. A Ru-complex (tris (2,2'-bipyridyl)ruthenium(II) chloride) was used as a fluorescent probe, and Egeria densa Planch. was used as a model aquatic plant. The DO-quenched fluorescence and material movement-induced deflection signals are compared at different distances from the aquatic plant surface. The results show that the optical detection system can monitor DO and the material movements at a vicinity of the aquatic plants not only much more sensitively, but also much more closely to real time than analytical methods that monitor concentration changes at a bulk solution.

Comparative Studies on Effects of Acid Solutions on Aquatic Plants by Beam Deflection and Absorbance Spectroscopy Methods.

The beam deflection method and absorbance spectroscopy were applied to study effects of acid solutions on aquatic plants, and their results were compared. Aquatic plants Egeria densa and Ceratophyllum demersum L were used as model plants. In absorbance experiments, a piece of the plants was put in a beaker with 20 mL HCl solution, and absorbance of the HCl solution was measured every 30 min. In beam deflection experiments, a probe beam from a He-Ne laser was focused to a vicinity of the plants in a culture dish with HCl solution by an objective lens, and deflection signals of the probe beam were monitored by a position sensor. Absorbance spectra of the HCl solutions with immersing of the plants showed absorbance below 410 nm, suggesting that some compounds leaked from the plants into the HCl solutions. Changes of absorbance and deflection signals with immersion time were examined for different pH levels. The changing trends of the absorbance and deflection signals with time were similar, but the absorbance changes were delayed for about 2 - 3 h. The absorbance method could not detect the effect of the pH 5.0 HCl solutions on the aquatic plants, while the deflection method could.

Real-time noninvasive monitoring of UV light-induced cell death by the deflection of a probe beam.

Cell death and its deregulation characterize numerous human diseases. Here, we report on real-time noninvasive monitoring of UV light-induced cell death by the deflection of a probe beam. UV light of 330-370 nm from a high-pressure Hg lamp illuminated cultured HepG2 cells, and at the same time a probe beam from a diode laser was passed through a vicinity of the HepG2 cell. The deflection signal of the probe beam, which was induced by changes of the concentration gradients in processes of the active materials movements across the cell membrane, was monitored. It was found that the deflection signals changed greatly after UV illumination, suggesting that the materials movements across the cell membrane were greatly affected by the UV illumination. After UV illumination of about 5400-7400 s at a light power of 0.028 W/cm(2), the deflection signals became little changed with time, suggesting that the living cells had been killed by the UV illumination. This conclusion agreed well with cell viability determinations of the traditional trypan blue method.

Direct sampling in air of capillary electrophoresis.

Capillary electrophoresis (CE) is usually off-line combined with an adsorption/desorption method to analyze gaseous or atmospheric samples. Here, we demonstrated direct sampling in the air of CE. The inlet end of a fused silica capillary filled with a pH 7.2 phosphate buffer was placed in the air for absorbing gaseous or volatile components, while the outlet end was immersed into a buffer vial at the low electric potential side. After a certain period of time, the inlet end was immersed into another buffer vial at the high electric potential side; CE was carried out by applying a high electric voltage of 20 kV. An UV-absorbance detector (wavelength, 254 nm) was used in CE. Evaporated vanillin in air was used as model gaseous sample. Experimental factors, such as a height difference between the inlet end and the outlet buffer, were investigated in detail. Fast detection of evaporated vanillin in indoor air was demonstrated.

Determination of vanillin in vanilla perfumes and air by capillary electrophoresis.

The present study investigated capillary electrophoretic detection of vanillin in vanilla perfume and air. An UV-absorbance detector was used in a home-made capillary electrophoretic instrument. A fused silica capillary (outer diameter: 364 µm, inner diameter: 50 µm) was used as a separation capillary, and a high electric voltage (20 kV) was applied across the two ends of the capillary. Total length of the capillary was 70 cm, and the effective length was 55 cm. Experimental results showed that the vanillin peak was detected at about 600, 450, and 500 seconds when pH of running buffers in CE were 7.2, 9.3, and 11.5, respectively. The peak area of vanillin was proportional to its concentration in the range of 0-10(-2) mol/L. The detection limit was about 10(-5) mol/L. Vanillin concentration in a 1% vanilla perfume sample was determined to be about 3×10(-4) mol/L, agreed well with that obtained by a HPLC method. Furthermore, determination of vanillin in air by combination of CE and active carbon adsorption method was investigated.

Comparison of chemiluminescence from luminol solution and luminol-TiO2 suspension after illumination of a 355 nm pulse laser.

Chemiluminescence (CL) from luminol solution and luminol-TiO2 suspension after illumination of a 355 nm pulse laser is compared. Both the CL systems showed the CL spectra with maximum wavelength of 430 nm, suggesting that the emission was from the excite state of 3-aminophthalate ion. The TiO2 photocatalytically induced luminol CL could be separately detected either when the pulse laser power was smaller than 0.15 mJ/pulse or a slit was placed beyond -2-2 mm in the vertical direction of the laser beam. The TiO2 photocatalytically induced luminol CL intensity was linear to the laser power, while that of the 355 nm pulse laser-induced was nonlinear. A log-log plot between the 355 nm pulse laser-induced luminol CL intensity and laser power showed a near-linear regression fit with a slope of 2.11, suggesting that a two-photon absorption process of luminol was present in the 355 nm pulse laser-induced luminol CL. Adsorbed oxygen on the surface of TiO2 seemed to greatly contribute to the photocatalytically induced CL.

Chemiluminescence from luminol solution after illumination of a 355 nm pulse laser.

Chemiluminescence (CL) on the time scale of microseconds to milliseconds from luminol solution after illumination of a 355 nm pulse laser is reported. It was found that the CL is the emission from 3-aminophthalate ion (AP*). In CL decay after the pulse laser illumination, a peak was observed from about 200 to 30 micros depending on the laser power and the luminol concentration. It seemed that there was a fast and slow decay process; their kinetics were greatly dependent on the laser power and the luminol concentration. Dissolved oxygen was involved in the CL and played the same role on the whole time scale of microseconds to milliseconds. Involvement of reactive oxygen species such as H(2)O(2), (1)O(2), O(2) (*-) and OH in the CL was examined by adding their scavengers. Experimental results suggested that the possibility of involvement of H(2)O(2) and (1)O(2) in the CL was low. The CL in time periods less than 50 micros might be related to *OH. The (*)O(2) (*-)-induced CL increased with time after 50 micros and became dominant on the time scale of milliseconds. The CL was considered to be caused by both the photoionization and type I reaction mechanisms.

Study of protein-protein binding reaction by whole-column fluorescence-imaged CIEF.

Whole-column fluorescence-imaged CIEF was applied to study protein-protein binding reaction. A homemade whole-column fluorescence-imaged CIEF experimental setup was built, and its CIEF performance was evaluated with native fluorescent protein green fluorescence protein and fluorescently labeled proteins (bovine albumin, human albumin, and BSA). pIs, focusing time, detection limits, and linear quantitative range of the proteins were obtained. Furthermore, the method was used to study FITC-protein A-human IgG binding reaction. Experimental results showed that the apparent binding ratio of the FITC-protein A to human IgG was 1:2, and pI of the binding conjugates were about 6.3-6.5. No binding reaction was found between green fluorescence protein and the fluorescent-labeled proteins.

On-capillary chemiluminescence detection for capillary electrophoresis with a single capillary.

On-capillary chemiluminescence detection for capillary electrophoresis with a single capillary was reported. A hole (about 30 microm diameter) was made on the capillary wall at about 50.5 cm from the inlet end. Hydrogen peroxide solution could enter the capillary from the hole, and mixed with luminol and copper(II) to produce chemiluminescence. The chemiluminescence was detected by a PMT under the hole. Several factors that influenced chemiluminescence intensity were investigated. The detection limits for luminol and N-(4-aminolbutyl)-N-ethylisoluminol (ABEI) were 1 x 10(-11) and 2 x 10(-10) mol L(-1), respectively. The method features simple construction and no dead volume.

Capillary electrophoresis with in-capillary solid-phase extraction sample cleanup.

An in-capillary, solid-phase extraction (SPE)-capillary electrophoresis (CE) method, with not only high preconcentration factor but also no adsorption on the inner capillary wall of absorbing species in real complex samples, has been developed with a hole-opened capillary. The SPE sorbents approximately 3 mm in length was packed in the inlet end of the capillary. A hole approximately 30 microm in diameter was opened after the sorbents on the capillary. Sample solutions were loaded from the inlet end, and the sample wastes flowed out from the hole. After a certain time of the sample loading, a 1.5-mm-long methanol plug was introduced from the inlet end and forced to pass by the sorbents and the hole. Then, a separation voltage was applied between the hole and the outlet end of the capillary to carry out normal CE. When the sample loading time was 120 min, CE peak heights of the 2,4-dichlorophenol and 2,4,5-trichlorophenol were proportional to their concentration in a range of 0.08-5 ng/mL, and their detection limits were 25 and 17 pg/mL, respectively. A 16,000-fold sensitivity enhancement was obtained for CE of the chlorophenols with only a little decrease in CE separation efficiency. It was also demonstrated with the mixture of the chlorophenols and a surfactant cetyltrimethylammonium bromide that the present method could eliminate the adsorption problem of absorbing species on the inner wall during sample loading. Furthermore, the SPE-CE was directly applied to determination of chlorophenols on the level of 0.02 ppb in downstream water of a river, and the results agreed well with those obtained with off-line SPE-HPLC experiments.

Hazard identification on a single cell level using a laser beam.

This research shows a novel method for hazard identification of a chemical and UV light on a single cell level with a laser probe beam. The laser probe beam was passed through interface of cell membrane/culture medium of a cultured human hepatoblastoma cell line HepG2. Deflection of the laser probe beam, which was induced by changes in concentration gradients due to the active materials movement across the cell membrane, was monitored. When a toxic hazard existed, a living cell was expected to be killed or injured, or cellular behaviors to be changed greatly. Then, the changing deflection signal from the living cell would become unchanged or altered in a different way. This was successfully demonstrated with cytotoxicity of UV light and H(2)O(2). Most of the cultured HepG2 cells showed changing deflection signals after 10 min illumination of UV-visible light longer than 370 nm, while almost all HepG2 cells showed unchanged deflection signal after 10 min illumination of UV-visible light with wavelength longer than 330 nm. The results suggested that UV light between 330-370 nm could kill the cells. Additions of H(2)O(2) solution with different concentrations to the cell cultures caused the changing deflection signal from a living cell either unchanged or changed in different trend, suggesting toxicity of H(2)O(2) to the cells. The results from the beam deflection detection agreed well with those obtained by the conventional trypane blue method.

Time-resolved chemiluminescence study of the TiO2 photocatalytic reaction and its induced active oxygen species.

The time-resolved chemiluminescence (CL) method has been applied to study the TiO(2) photocatalytic reaction on a micros-ms timescale. The experimental set-up for time-resolved CL was improved for confirmation of the unique luminol CL induced by the photocatalytic reaction. The third harmonic light (355 nm) from an Nd:YAG laser was used for the light source of the TiO(2) photocatalytic reaction. Luminol CL induced by this reaction was detected by a photomultiplier tube (PMT) and a preamplifier was used for amplifying the CL signal. Experimental conditions affecting the photocatalytically induced CL were discussed in detail. The involvement of active oxygen species such as .OH, O(2) (.-) and H(2)O(2) in the CL was examined by adding their scavengers. It is concluded that .OH was greatly involved in the CL on a micros-ms timescale, especially in time periods <100 micros after illumination of the pulse laser. On the other hand, CL generated by O(2) (.-) began to increase after 100 micros and became dominant after 2.5 ms. A small part of the CL might be generated by H(2)O(2) on the whole micros-ms timescale. A CL reaction mechanism related with .OH and dissolved oxygen was proposed to explain the photocatalytically induced luminol CL on a micros-ms timescale, especially in periods <100 micros.

In-capillary solid-phase extraction-capillary electrophoresis for the determination of chlorophenols in water.

A novel CE method combined with SPE in a single capillary was developed for analysis of chlorophenols in water. A frit of 0.5 mm was first made by a sol-gel method, followed by packing a SPE sorbent in the inlet end of the capillary. Two phenol derivatives, 2,4-dichlorophenol and 2,4,5-trichlorophenol, were used as the model compounds. By loading sample solutions into the capillary, the two chlorophenols were extracted into the sorbent. They were desorbed by injecting only about 4 nL of methanol. Finally, the analytes were separated by conventional CE. The technique provided a concentration enhancement factor of over 4000-fold for both chlorophenols. The detection limits (S/N = 3) of 2,4-dichlorophenol and 2,4,5-trichlorophenol were determined to be 0.1 ng/mL and 0.07 ng/mL, respectively. For replicate analyses of 5 ng/mL of 2,4-dichlorophenol, within-day and between-day RSDs of migration time, peak height and peak area were in the range of 1.8-2.0%, 4.0-4.4% and 4.1-4.6%, respectively. The method shows wide linear range, acceptable reproducibility and excellent sensitivity, and it was applied to the analyses of spiked river water samples. The capillary packed with the SPE sorbents can be used for more than 400 runs without performance deterioration.

Isoelectric focusing sample injection for capillary zone electrophoresis in a fused silica capillary.

An improvement has been made to couple isoelectric focusing (IEF) sample injection and capillary zone electrophoresis in an untreated fused silica capillary. Electroosmotic flow is efficiently prevented by simply using a rubber block at the outlet end of the capillary during IEF sample injection. The experimental conditions that affect the concentration effect are discussed. A concentration enhancement factor of over 100-fold can be easily obtained for two model proteins: lysozyme and ribonuclease A.

Noninvasive diagnosis of a single cell with a probe beam.

It is important to distinguish a living/dead cell in cell culture, especially in the regenerate medicine field including cell therapy, since those cells are usually in short supply and consequently the ex vivo culture process should be operated strictly. Conventional methods for distinguishing a living from a dead cell usually require labeling with a dye, which spoils the culture of the cell. Here we show a simple noninvasive method for diagnosing a dead or alive cell with a probe beam. If a cell is alive, the active transport of materials across the cell membrane causes a change of concentration gradients, and this change further induces a change of deflection of a probe beam passing through a vicinity of the cell membrane. If a cell is dead, no or little change in deflection of the probe beam is induced because no or little active materials movement across the cell membrane exists. The deflection of the probe beam is monitored, and judgment on whether a cell is dead or alive from the deflection signal agreed with the conventional decision.

In-capillary preconcentration of proteins for capillary electrophoresis using a cellulose acetate-coated porous joint.

This work describes the in-capillary preconcentration of proteins using a cellulose acetate-coated porous joint. The capillary wall near the inlet end of a capillary was made porous by HF etching. During the etching process, a voltage was applied across the capillary wall and the electric current across it was monitored. As the current passed through the capillary wall, it became porous. A solution of cellulose acetate in acetone was added to the etched porous joint. After the acetone was evaporated off, a cellulose acetate-coated porous joint was formed. To preconcentrate the protein ions, an electric voltage was applied between the inlet end of the capillary and the coated porous joint; the protein ions electromigrated to the porous joint but could not pass through it, while the buffer ions could pass easily through the joint. After allowing a certain amount of time for protein preconcentration, a separation voltage was applied across the two ends of the capillary, and normal capillary electrophoresis was carried out. The preconcentration factors for cytochrome c, lysozyme, ribonuclease, and chymotrypsinogen were 65, 155, 705, and 800, respectively. The cellulose acetate-coated porous joint was shown to be strong and stable over time, and was used to analyze trace proteins and macromolecules in biological samples.

Isoelectric focusing sample injection for capillary electrophoresis of proteins.

Carrier ampholyte-free isoelectric focusing (IEF) sample injection (concentration) for capillary electrophoresis (CE) is realized in a single capillary. A short section of porous capillary wall was made near the injection end of a capillary by HF etching. In the etching process, an electric voltage was applied across the etching capillary wall and electric current was monitored. When an electric current through the etching capillary was observed, the capillary wall became porous. The etched part was fixed in a vial, where NaOH solution with a certain concentration was added during the sample injection. The whole capillary was filled with pH 3.0 running buffer. The inlet end vial was filled with protein sample dissolved in the running buffer. An electric voltage was applied across the inlet end vial and etched porous wall. A neutralization reaction occurs at the boundary (interface) of the fronts of H+ and OH-. A pH step or sharp pH gradient exists across the boundary. When positive protein ions electromigrate to the boundary from the sample vial, they are isoelectricelly focused at points corresponding to their pH. After a certain period of concentration, a high voltage is applied across the whole capillary and a conventional CE is followed. An over 100-fold concentration factor has been easily obtained for three model proteins (bovine serum albumin, lysozyme, ribonuclease A). Furthermore, the IEF sample concentration and its dynamics have been visually observed with the whole-column imaging technique. Its merits and remaining problem have been discussed, too.

Chemiluminescence study of active oxygen species produced by TiO2 photocatalytic reaction.

Two chemiluminescence approaches have been used for study of active oxygen species produced by the TiO2 photocatalytic reaction. One is based on flow injection analysis (FIA)-luminol chemiluminescence (CL); another is a time-resolved CL method. In the FIA-CL experiment, an UV-illuminated TiO2 suspension and water were passed into a mixing cell by two separate flow lines. Luminol solution was injected into the water flow line at different times. The injected luminol reacted with active oxygen species generated by the TiO2 photocatalytic reaction in a mixing coil and produced CL. It was found that the maximum CL was detected at the first injection of luminol. CL intensity decreased with time of injection. When the luminol was injected after 5 min, the CL intensity was almost unchanged. Addition of scavengers of active oxygen species indicated that the CL produced early in the 5 min was caused by O2- and H2O2, while CL after 5 min was only from H2O2. In the time-resolved CL, the third harmonic wavelength of Nd:YAG laser (355 nm) was used as a UV light source, and CL was detected by a PMT and recorded in a millisecond time scale using a digital oscilloscope. It was found that CL induced by the photocatalytic reaction increased with concentration of the TiO2 suspension. Scavengers of active oxygen species of *OH, O2- and H2O2 were added to study the involvement of the active oxygen species.