Analysis of individual mitochondria via fluorescent immunolabeling with Anti-TOM22 antibodies

Mitochondria are responsible for maintaining a variety of cellular functions. One such function is the interaction and subsequent import of proteins into these organelles via the translocase of outer membrane (TOM) complex. Antibodies have been used to analyze the presence and function of proteins comprising this complex, but have not been used to investigate variations in the abundance of TOM complex in mitochondria. Here, we report on the feasibility of using capillary cytometry with laser-induced fluorescence to detect mitochondria labeled with antibodies targeting the TOM complex and to estimate the number of antibodies that bind to these organelles. Mitochondria were fluorescently labeled with DsRed2, while antibodies targeting the TOM22 protein, one of nine proteins comprising the TOM complex, were conjugated to the Atto-488 fluorophore. At typical labeling conditions, 94 % of DsRed2 mitochondria were also immunofluorescently labeled with Atto-488 Anti-TOM22 antibodies. The calculated median number of Atto-488 Anti-TOM22 antibodies bound to the surface of mitochondria was ～2,000 per mitochondrion. The combination of fluorescent immunolabeling and capillary cytometry could be further developed to include multicolor labeling experiments, which enable monitoring several molecular targets at the same time in the same or different organelle types.

Monitoring subcellular biotransformation of N-l-leucyldoxorubicin by micellar electrokinetic capillary chromatography coupled to laser-induced fluorescence detection

Development of prodrugs is a promising alternative to address cytotoxicity and nonspecificity of common anticancer agents. N-l-leucyldoxorubicin (LeuDox) is a prodrug that is biotransformed to the anticancer drug doxorubicin (Dox) in the extracellular space; however, its biotransformation may also occur intracellularly in endocytic organelles. Such organelle-specific biotransformation is yet to be determined. In this study, magnetically enriched endocytic organelle fractions from human uterine sarcoma cells were treated with LeuDox. Micellar electrokinetic chromatography with laser-induced fluorescence detection (MEKC-LIF) was used to determine that 10 % of LeuDox was biotransformed to Dox, accounting for ～43 % of the biotransformation occurring in the post-nuclear fraction. This finding suggests that endocytic organelles also participate in the intracellular biotransformation of LeuDox to Dox.

Glassy carbon electrode modified with poly-Neutral Red for photoelectrocatalytic oxidation of NADH

A new approach is described for the photoelectrocatalytic oxidation of Reduced ß-Nicotinamide Adenine Dinucleotide (NADH). It is based on a glassy carbon electrode (GCE) modified with a film of poly-Neutral Red (poly-NR) that is obtained by electropolymerization. Electrochemical measurements revealed that the modified electrode displays electrocatalytic and photo-electrocatalytic activity towards oxidation of NADH. If irradiated with a 250-W halogen lamp, the electrode yields a strongly increased electrocatalytic current compared to the current without irradiation. Amperometric and photo-amperometric detection of NADH was performed at +150 mV vs. Ag/AgCl/KCl_sat and the currents obtained are linearly related to the concentration of NADH. Linear calibration plots are obtained in the concentration range from 1.0 μM to 1.0 mM for both methods. However, the slope of the current-NADH concentration curve of the photo-electrocatalytic procedure was 2-times better than that obtained without irradiation.

Albumin coated CuInS2 quantum dots as a near-infrared fluorescent probe for NADH, and their application to an assay for pyruvate

Water-soluble CuInS₂ quantum dots (QDs) stabilized with 3-mercaptopropionic acid were synthesized in aqueous solution and then coated with bovine serum albumin. The resulting particles display fluorescence with a peak at 680 nm that is effectively quenched by 1, 4-dihydro-nicotinamide adenine dinucleotide (NADH), but not by 1, 4-nicotinamide adenine dinucleotide (NAD⁺). The enzyme lactate dehydrogenase catalyzes the reduction of pyruvate and dehydrogenation of lactic acid using NAD⁺ or NADH as a cosubstrate. The new QDs were applied to monitor the course of lactate dehydrogenase-catalyzed reaction of pyruvate by detecting NADH via its quenching effect. This resulted in a convenient and selective detection scheme for pyruvate. The detection limit is as low as 25 nM.

Glucose biosensor based on glucose oxidase immobilized on unhybridized titanium dioxide nanotube arrays

A glucose biosensor has been fabricated by immobilizing glucose oxidase (GOx) on unhybridized titanium dioxide nanotube arrays using an optimized cross-linking technique. The TiO₂ nanotube arrays were synthesized directly on a titanium substrate by anodic oxidation. The structure and morphology of electrode material were characterized by X-ray diffraction and scanning electron microscopy. The electrochemical performances of the glucose biosensor were conducted by cyclic voltammetry and chronoamperometry measurements. It gives a linear response to glucose in the 0.05 to 0.65 mM concentration range, with a correlation coefficient of 0.9981, a sensitivity of 199.6 μA mM^?1 cm^?2, and a detection limit as low as 3.8 µM. This glucose biosensor exhibited high selectivity for glucose determination in the presence of ascorbic acid, sucrose and other common interfering substances. This glucose biosensor also performed good reproducibility and long-time storage stability. This optimized cross-linking technique could open a new avenue for other enzyme biosensors fabrication.

Highly sensitive sensor for detection of NADH based on catalytic growth of Au nanoparticles on glassy carbon electrode

In this work, an electrochemical dihydronicotinamide adenine dinucleotide (NADH) sensor based on the catalytic growth of Au nanoparticles (Au NPs) on glassy carbon electrode was developed. Catalyzed by Au NPs immobilized on pretreated glassy carbon electrode, the reduction of AuCl₄ ^? in the presence of hydroquinone and cetyltrimethyl ammonium chloride led to the formation of enlarged Au NPs on the electrode surface. Spectrophotometry and high-resolution scanning electronic microscope (SEM) analysis of the sensor morphologies before and after biocatalytic reaction revealed a diameter growth of the nanoparticles. The catalytic growth of Au NPs on electrode surface remarkably facilitated the electron transfer and improved the performance of the sensor. Under optimal conditions, NADH could be detected in the range from 1.25?×?10^?6 to 3.08?×?10^?4 M, and the detection limit was 2.5?×?10^?7 M. The advantages of the proposed sensor, such as high precision and sensitivity, fast response, low cost, and good storage stability, made it suitable for on-line detection of NADH in complex biological systems and contaminant degradation processes.

Large scale biomimetic membrane arrays

To establish planar biomimetic membranes across large scale partition aperture arrays, we created a disposable single-use horizontal chamber design that supports combined optical–electrical measurements. Functional lipid bilayers could easily and efficiently be established across CO₂ laser micro-structured 8?×?8 aperture partition arrays with average aperture diameters of 301?±?5 μm. We addressed the electro-physical properties of the lipid bilayers established across the micro-structured scaffold arrays by controllable reconstitution of biotechnological and physiological relevant membrane peptides and proteins. Next, we tested the scalability of the biomimetic membrane design by establishing lipid bilayers in rectangular 24?×?24 and hexagonal 24?×?27 aperture arrays, respectively. The results presented show that the design is suitable for further developments of sensitive biosensor assays, and furthermore demonstrate that the design can conveniently be scaled up to support planar lipid bilayers in large square-centimeter partition arrays.

VIZR—an automated chemometric technique for metabolic profiling

A chemometric technique, visual interpretation of z-score ratios (VIZR), written in the open source code R, has been developed to identify metabolic differences between individual biosamples and a control group. To demonstrate the capabilities of VIZR, 49 urine samples were collected from healthy volunteers: 41 samples were collected randomly following a normal dietary routine and 7 test samples were collected after dietary supplementation with either ibuprofen or alcoholic beverages. An eighth test sample was prepared by 50 % dilution of a control sample. Sample analysis was conducted by ¹H nuclear magnetic resonance (NMR) spectroscopy and the collected data were subjected to VIZR analysis, which successfully discriminated each of the 8 test samples from the 41 control samples. In addition, VIZR analysis revealed the NMR spectral regions responsible for the disparity between the individual test samples and the control group. The self-normalizing nature of the VIZR calculation provides a robust analysis independent of dilution effects, which is especially important in urine analyses. Potential applications of VIZR include high-throughput data analysis for toxicological profiling, disease diagnosis, and biomarker identification in any type of biosample for which a control dataset can be established. Although demonstrated herein for the statistical analysis of ¹H NMR data, the VIZR program is platform independent and could be applied to digitized metabolic datasets acquired using other techniques including hyphenated mass spectrometry measurements.

A microfluidic system to study the cytotoxic effect of drugs: the combined effect of celecoxib and 5-fluorouracil on normal and cancer cells

We have investigated the response of normal and cancer cells to exposure a combination of celecoxib (Celbx) and 5-fluorouracil (5-FU) using a lab-on-a-chip microfluidic device. Specifically, we have tested the cytotoxic effect of Celbx on normal mouse embryo cells (Balb/c 3T3) and human lung carcinoma cells (A549). The single drugs or their combinations were adjusted to five different concentrations using a concentration gradient generator (CGG) in a single step. The results suggest that Celbx can enhanced the anticancer activity of 5-FU by stronger inhibition of cancer cell growth. We also show that the A549 cancer cells are more sensitive to Celbx than the Balb/c 3T3 normal cells. The results obtained with the microfluidic system were compared to those obtained with a macroscale in vitro cell culture method. In our opinion, the microfluidic system represents a unique approach for an evaluation of cellular response to multidrug exposure that also is more simple than respective microwell plate assays.

Capillary electrophoretic analysis of hydroxyl radicals produced by respiring mitochondria

Here, we report the use of a capillary electrophoretic method with laser-induced fluorescence detection to evaluate hydroxyl radicals produced by respiring mitochondria. The probe, hydroxyphenylfluorescein (HPF), is separated from the product, fluorescein, in under 5 min with zeptomole and attomole limits of detection for fluorescein and HPF, respectively. Purification of the probe with a C-18 SPE column is necessary to reduce the fluorescein impurity in the probe stock solution from 0.4 % to less than 0.001 %. HPF was responsive to hydroxyl radicals produced by isolated mitochondria from L6 cells, and this signal was blunted when DMSO was added to scavenge hydroxyl radicals and when carbonyl cyanide m-chlorophenylhydrazone was added to depolarize the mitochondria. The method was used to compare hydroxyl radical levels in mitochondria isolated from brown adipose tissue of lean and obese mice. Mitochondria from obese mice produced significantly more hydroxyl radicals than those from lean mice.

Patterned growth of vertically aligned silicon nanowire arrays for label-free DNA detection using surface-enhanced Raman spectroscopy

Patterning is of paramount importance in many areas of modern science and technology. As a good candidate for novel nanoscale optoelectronics and miniaturized molecule sensors, vertically aligned silicon nanowire (SiNW) with controllable location and orientation is highly desirable. In this study, we developed an effective procedure for the fabrication of vertically aligned SiNW arrays with micro-sized features by using single-step photolithography and silver nanoparticle-induced chemical etching at room temperature. We demonstrated that the vertically aligned SiNW arrays can be used as a platform for label-free DNA detection using surface-enhanced Raman spectroscopy (SERS), where the inherent “fingerprint” SERS spectra allows for the differentiation of closely related biospecies. Since the SiNW array patterns could be modified by simply varying the mask used in the photolithographic processing, it is expected that the methodology can be used to fabricate label-free DNA microarrays and may be applicable to tissue engineering, which aims to create living tissue substitutes from cells seeded onto 3D scaffolds.

Detection of catecholamines in single specimens of groundwater amphipods

Catecholamines play essential roles in several physiological processes in vertebrates as well as in invertebrates. While several studies have shown the presence of these substances in surface water invertebrates, their occurrence in groundwater fauna is unproven. In the present study, the presence of different catecholamines (i.e., noradrenaline, adrenaline, and dopamine) in individual specimens of groundwater amphipods of the genus Niphargus (mostly Niphargus inopinatus) was investigated via two independent analytical methods: HPLC/EcD and UPLC/TOF-MS. Mean values for catecholamine levels were 533 pg mg^?1 fresh weight for noradrenaline, 314 pg mg^?1 for adrenaline, and 16.4 ng mg^?1 for dopamine. The optimized protocol allowed the detection of CAs in single organisms of less than 1 mg fresh weight. Catecholamine concentration patterns in groundwater invertebrates are briefly discussed here with respect to their evolutionary adaptation to an environmentally stable, energy-poor habitat.

REMUS100 AUV with an integrated microfluidic system for explosives detection

Quantitating explosive materials at trace concentrations in real-time on-site within the marine environment may prove critical to protecting civilians, waterways, and military personnel during this era of increased threat of widespread terroristic activity. Presented herein are results from recent field trials that demonstrate detection and quantitation of small nitroaromatic molecules using novel high-throughput microfluidic immunosensors (HTMI) to perform displacement-based immunoassays onboard a HYDROID REMUS100 autonomous underwater vehicle. Missions were conducted 2–3 m above the sea floor, and no HTMI failures were observed due to clogging from biomass infiltration. Additionally, no device leaks were observed during the trials. HTMIs maintained immunoassay functionality during 2 h deployments, while continuously sampling seawater absent without any pretreatment at a flow rate of 2 mL/min. This 20-fold increase in the nominal flow rate of the assay resulted in an order of magnitude reduction in both lag and assay times. Contaminated seawater that contained 20–175 ppb trinitrotoluene was analyzed.

Green biosynthesis of silver nanoparticles and nanomolar detection of p-nitrophenol

Green biosynthesis of nanoparticles and their applications in sensor field is of great interest to the researchers. We report herein a simple green approach for the synthesis of silver nanoparticles (Ag-NPs) using Acacia nilotica Willd twig bark and its application for the detection of 4-nitro phenol (4-NP). The synthesized Ag-NPs were characterized by Transmission electron microscopy, X-ray diffraction and elemental analysis. The size of synthesized Ag-NPs was in the range of 10–50 nm. The Ag-NPs modified electrode shows a high sensitivity and selectivity towards the sensing of 4-NP. The fabricated modified electrode shows a low detection limit of 15 nM on the wider linear response range from 100 nM to 350 μM with the sensitivity of 2.58?±?0.05 μAμM^?1 cm^?2. In addition, the fabricated sensor shows good repeatability and reproducibility.

Poly(ethylene oxide)–poly(ethylene imine) block copolymers as templates and catalysts for the in situ formation of monodisperse silica nanospheres

Block copolymers based on poly(ethylene oxide) (PEO) and poly(ethylene imine) (PEI) are efficient catalysts/templates for the formation of uniform silica nanoparticles. Addition of tetraethylorthosilicate to a solution of PEO–PEI or PEI–PEO–PEI block copolymers results in the formation of silica particles with a diameter of ca. 30 nm and narrow size distribution. The particles precipitated with the diblock copolymers can be redispersed in water after isolation as individual nanoparticles. Evidently, block copolymers based on PEO and PEI serve as excellent templates for the biomimetic and “soft” synthesis of silica nanoparticles.

Highly sensitive uric acid biosensor based on individual zinc oxide micro/nanowires

We describe the use of individual zinc oxide (ZnO) micro/nanowires in an electrochemical biosensor for uric acid. The wires were synthesized by chemical vapor deposition and possess uniform morphology and high crystallinity as revealed by scanning electron microscopy, X-ray diffraction, and photoluminescence studies. The enzyme uricase was then immobilized on the surface of the ZnO micro/nanowires by physical adsorption, and this was proven by Raman spectroscopy and fluorescence microscopy. The resulting uric acid biosensor undergoes fast electron transfer between the active site of the enzyme and the surface of the electrode. It displays high sensitivity (89.74 μA cm^?2 mM^?1) and a wide linear analytical range (between 0.1 mM and 0.59 mM concentrations of uric acid). This study also demonstrates the potential of the use of individual ZnO micro/nanowires for the construction of highly sensitive nano-sized biosensors.

Metabolic pathway elucidation towards time- and dose-dependent electrophoretic screening of stable oxidative phenolic compounds

This study demonstrates an untested link between model phenolic compounds and the formation/electrophoretic separation of stable urinary metabolites. Sterically encumbered carbonyl groups were examined, and mass determination was used to confirm the presence and stability of two oxidative metabolites of pentachlorophenol: tetrachloro-1,2-benzoquinone and tetrachloro-1,4-dihydroquinone. Subsequently, baseline resolved separation of pentachlorophenol and the two oxidative metabolites was demonstrated under the following conditions: 75 mM sodium tetraborate buffer (pH?=?8.5) with 5 % methanol and 50 mM SDS, +10.0 kV running voltage, injection time?=?5.0 s, effective capillary length?=?55 cm, and run temperature?=?20 °C. Results not only provide key metabolic inferences for pentachlorophenol, they also exhibit improvements in the ability to separate and detect changes in urinary metabolites in response to phenolic-related exposure.

Chemical imaging of trichome specialized metabolites using contact printing and laser desorption/ionization mass spectrometry

Cell transfer by contact printing coupled with carbon-substrate-assisted laser desorption/ionization was used to directly profile and image secondary metabolites in trichomes on leaves of the wild tomato Solanum habrochaites. Major specialized metabolites, including acyl sugars, alkaloids, flavonoids, and terpenoid acids, were successfully detected in positive ion mode or negative ion mode, and in some cases in both modes. This simple solvent-free and matrix-free sample preparation for mass spectrometry imaging avoids tedious sample preparation steps, and high-spatial-resolution images were obtained. Metabolite profiles were generated for individual glandular trichomes from a single Solanum habrochaites leaf at a spatial resolution of around 50 μm. Relative quantitative data from imaging experiments were validated by independent liquid chromatography–mass spectrometry analysis of subsamples from fresh plant material. The spatially resolved metabolite profiles of individual glands provided new information about the complexity of biosynthesis of specialized metabolites at the cellular-resolution scale. In addition, this technique offers a scheme capable of high-throughput profiling of metabolites in trichomes and irregularly shaped tissues and spatially discontinuous cells of a given cell type.

157 nm Photodissociation of Dipeptide Ions Containing N-Terminal Arginine

Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m₂ and m₂+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.

Combination of UHPLC/Q-TOF-MS,NMR spectroscopy,and ECD calculation for screening and identification of reactive metabolites of gentiopicroside in humans

The metabolic investigation of natural products is a great challenge because of unpredictable metabolic pathways, little knowledge on metabolic effects, and lack of recommended analytical methodology. Herein, a combined strategy based on ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF-MS), nuclear magnetic resonance (NMR) spectroscopy, and electronic circular dichroism (ECD) calculation was developed and employed for the human metabolism study of gentiopicroside (GPS), a naturally hepato-protective iridoid glycoside. The whole metabolic study consisted of three major procedures. First, an improved UHPLC/Q-TOF-MS method was used to separate and detect a total of 15 GPS metabolites that were obtained from urine samples (0 to 72 h) of 12 healthy male participants after a single 50-mg oral dose of GPS. Second, a developed “MS-NMR-MS” method was applied to accurately identify molecular structures of the observed metabolites. Finally, given that the associated stereochemistry may be a crucial factor of the metabolic activation, the absolute configuration of the reactive metabolites was revealed through chemical calculations. Based on the combined use, a pair of diastereoisomers (G05 and G06) were experimentally addressed as the bioreactive metabolites of GPS, and the stereochemical determination was completed. Whereas several novel metabolic transformations, occurring via oxidation, N-heterocyclization and glucuronidation after deglycosylation, were also observed. The results indicated that GPS has to undergo in vivo metabolism-based activation to generate reactive molecules capable of processing its hepato-protective activity.