Miniaturization of environmental chemical assays in flowing systems: the lab-on-a-valve approach vis-à-vis lab-on-a-chip microfluidic devices

The analytical capabilities of the microminiaturized lab-on-a-valve (LOV) module integrated into a microsequential injection (μSI) fluidic system in terms of analytical chemical performance, microfluidic handling and on-line sample processing are compared to those of the micro total analysis systems (μTAS), also termed lab-on-a-chip (LOC). This paper illustrates, via selected representative examples, the potentials of the LOV scheme vis-à-vis LOC microdevices for environmental assays. By means of user-friendly programmable flow and the exploitation of the interplay between the thermodynamics and the kinetics of the chemical reactions at will, LOV allows accommodation of reactions which, at least at the present stage, are not feasible by application of microfluidic LOC systems. Thus, in LOV one may take full advantage of kinetic discriminations schemes, where even subtle differences in reactions are utilized for analytical purposes. Furthermore, it is also feasible to handle multi-step sequential reactions of divergent kinetics; to conduct multi-parametric determinations without manifold reconfiguration by utilization of the inherent open-architecture of the micromachined unit for implementation of peripheral modules and automated handling of a variety of reagents; and most importantly, it offers itself as a versatile front end to a plethora of detection schemes. Not the least, LOV is regarded as an emerging downscaled tool to overcome the dilemma of LOC microsystems to admit real-life samples. This is nurtured via its intrinsic flexibility for accommodation of sample pre-treatment schemes aimed at the on-line manipulation of complex samples. Thus, LOV is playing a prominent role in the environmental field, whenever the monitoring of trace level concentration of pollutants is pursued, because both matrix isolation and preconcentration of target analytes is most often imperative, or in fact necessary, prior to sample presentation to the detector.     

A fast and highly sensitive detection of cholesterol using polymer microfluidic devices and amperometric system

In this work, the rapid detection of cholesterol using poly(dimethylsiloxane) microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, was developed. Direct amperometric detection for poly(dimethylsiloxane) (PDMS) microchip capillary electrophoresis was successfully applied to quantify cholesterol levels. Factors influencing the performance of the method (such as the concentration and pH value of buffer electrolyte, concentration of cholesterol oxidase enzyme (ChOx), effect of solvent on the cholesterol solubility, and interferences) were carefully investigated and optimized. The migration time of hydrogen peroxide, product of the reaction, was less than 100 s when using 40 mM phosphate buffer at pH 7.0 as the running buffer, a concentration of 0.68 U/mL of the ChOx, a separation voltage of +1.6 kV, an injection time of 20 s, and a detection potential of +0.5 V. PDMS microchip capillary electrophoresis showed linearity between 38.7 μg/dL (1 μM) and 270.6 mg/dL (7 mM) for the cholesterol standard; the detection limit was determined as 38.7 ng/dL (1 nM). To demonstrate the potential of this assay, the proposed method was applied to quantify cholesterol in bovine serum. The percentages of recoveries were assessed over the range of 98.9-101.8%. The sample throughput was found to be 60 samples per hour. Therefore, PDMS microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, is very rapid, accurate and sensitive method for the determination of cholesterol levels.     

Nanomolar detection with high sensitivity microfluidic absorption cells manufactured in tinted PMMA for chemical analysis

We describe a novel, cost effective and simple technique for the manufacture of high sensitivity absorption cells for microfluidic analytical systems. The cells are made from tinted polymethyl methacrylate (PMMA) in which microfluidic channels are fabricated. Two windows (typically 250 μm thick, resulting in little optical power loss) are formed at either end of the channel through which light is coupled. Unwanted stray light from the emitter passes through a greater thickness of the tinted substrate (typically the length of the cell) and is preferentially absorbed. In effect, this creates a pin-hole configuration over the length of the absorption cell, providing improved performances (sensitivity, S/N ratios, baseline noise and limit of detection) when used as an absorption cell compared to clear substrates. The method is used to achieve a LOD of 20 nM with a colourimetric iron assay and a LOD of 0.22 milli-absorption units with a pH assay.     

Synthesis and anticancer study of 9-aminoacridine derivatives

Acridine and its derivatives, well known as DNA intercalates lead to cell cycle arrest and apoptosis. 9-Aminoacridine derivatives were synthesized, characterized and evaluated against lung cancer (A-549) cell line and cervical cancer (HeLa) cell line by MTT assay. Compound 9 exhibited potent anticancer activity with CTC₅₀ (13.75 & 18.75 μg/ml) for cervical cancer cell (HeLa) line and lung cancer cell (A-549) line respectively. In vitro short term cytotoxicity evaluation of compound 9 was carried out by Dalton’s Lymphoma Ascites (DLA) with percentage growth inhibition CTC₅₀ (337.5 μg/ml). Compound 7 also exhibited good anticancer activity with CTC₅₀ (31.25 & 36.25 μg/ml) for cervical cancer cell (HeLa) line and lung cancer cell (A-549) line respectively. Further in vivo study of newly synthesized 9-aminoacridine derivative can give a ray of light in the field of anticancer drugs.     

Synthesis and biological evaluation of new 9-aminoacridine-4-carboxamide derivatives as anticancer agents: 1st Cancer Update

The design of molecules that recognize the specific sequence of the DNA double helix or those that can stabilize DNA topoisomerase cleavable complex to stop the progression of DNA process, may be very useful in cancer chemotherapy. In the field of antituor DNA-intercalating agents, 9-aminoacridine-4-carboxamide derivatives play an important role due to their anti-proliferative properties. In the present study, 9-aminoacridine-4-carboxamide derivatives were designed, synthesized, characterized and evaluated against lung cancer (A-549) cell line and cervical cancer (HeLa) cell line in vitro by MTT assay. Compounds 5a, 5b and 5e were selected for anticancer evaluation against the lung cancer cell line and cervical cancer cell line. Compound 5b showed the maximum activity against the cervical cancer (HeLa) cell line with CTC₅₀ (47.50 μg/ml) and compound 5e showed the maximum activity against the lung cancer (A-549) cell line with CTC₅₀ (100 μg/ml) among the tested compounds. The present study opens new vista for DNA intercalating anticancer compounds and their further in vivo investigation.     

Toward Multitasking Pharmacological COX-Targeting Agents: Non-Steroidal Anti-Inflammatory Prodrugs with Antiproliferative Effects

The antitumor activity of certain anti-inflammatory drugs is often attributed to an indirect effect based on the inhibition of COX enzymes. In the case of anti-inflammatory prodrugs, this property could be attributed to the parent molecules with mechanism other than COX inhibition, particularly through formulations capable of slowing down their metabolic conversion. In this work, a pilot docking study aimed at comparing the interaction of two prodrugs, nabumetone (NB) and its tricyclic analog 7-methoxy-2,3-dihydro-1H-cyclopenta[b]naphthalen-1-one (MC), and their common active metabolite 6-methoxy-2-naphthylacetic acid (MNA) with the COX binding site, was carried out. Cytotoxicity, cytofluorimetry, and protein expression assays on prodrugs were also performed to assess their potential as antiproliferative agents that could help hypothesize an effective use as anticancer therapeutics. Encouraging results suggest that the studied compounds could act not only as precursors of the anti-inflammatory metabolite, but also as direct antiproliferative agents.     

A novel carbazole-based ratiometric fluorescent sensor for Zn recognition through excimer formation and application of the resultant complex for colorimetric recognition of oxalate through IDAs

A new carbazole-based Zn²⁺ selective fluorescent sensor L has been developed. In CH₃CN/H₂O (1:1, v/v, HEPES 10 mM, pH=7.4) solution, L displays selective and ratiometric responses to Zn²⁺ through excimer formation. The Zn²⁺ recognition process has good anti-interference ability over other metal ions. The dinuclear complex Zn₂L₂ was further used as a receptor for oxalate. Through constructing a chemosensing ensemble with chromeazurol S, colorimetric recognition of oxalate in water solution was achieved via indicator displacement assays. The oxalate recognition process exhibits obvious color changes from blue to yellow and is naked eye detectable.     

Expanding the scope of MS binding assays to low-affinity markers as exemplified for mGAT1

Following a recently developed concept of MS binding assays based on the quantification of a native marker by LC–MS a procedure to study binding of a low-affinity marker in kinetic, saturation, and competition experiments was established. Separation of bound and unbound marker—the most crucial step of the assay—could be effectively achieved by filtration in a 96-well-format. MS binding assays according to this procedure allowed the reliable characterization of NO 711 binding to mGAT1 in presence of physiological NaCl concentrations. Comparing the results obtained in the present study with those from experiments using 1 mol L⁻¹ NaCl in the incubation milieu reveals remarkable differences with respect to the marker’s affinity and kinetics and to the investigated test compound’s potency. Principle of MS binding assays After incubation of a target with a native marker, bound and unbound marker are separated by filtration. Subsequently, the bound native marker is liberated from the target and finally quantified by LC-MS-MS. Dedicated to Prof. Hans-Dietrich Stachel on the occasion of his 80th birthday     

Synthesis,spectroscopic characterization,in vitro cytotoxic and structure activity relationships of some mononuclear Ru(II) complexes

New mononuclear Ru(II) complexes [Ru(A)₂(B)]²⁺, where A?=?2,2′-bipyridine/1,10-phenanthroline and B?=?3,4,5-tri-OCH₃-DPC, 4-CH₃-DPC, 4-N(CH₃)₂-DPC, 4-NO₂-DPC, N-BITSZ, PTSZ and PINH, were prepared and characterized by spectroscopic methods. The in vitro cytotoxic activities of the complexes and their corresponding ligands were investigated against the human cancer T-lymphocyte cell lines molt 4/c8 and CEM and the murine tumor leukemia cell line L1210, human promyelocytic leukemia cells (HL-60) and Bel-7402 liver cancer cells by MTT assay. The complexes [Ru(A)₂(B)]²⁺ (A?=?1,10-phenanthroline, B?=?3,4,5-tri-OCH₃-DPC) exerts rather more potent activities against all of these cell lines, especially for CEM and L1210. Ru complexes and structure–activity relationships and anticancer mechanisms are also discussed.     

A homogeneous assay for protein analysis in droplets by fluorescence polarization

We present a novel homogeneous (“mix‐incubate‐read”) droplet microfluidic assay for specific protein detection in picoliter volumes by fluorescence polarization (FP), for the first time demonstrating the use of FP in a droplet microfluidic assay. Using an FP‐based assay we detect streptavidin concentrations as low as 500 nM and demonstrate that an FP assay allows us to distinguish droplets containing 5 μM rabbit IgG from droplets without IgG with an accuracy of 95%, levels relevant for hybridoma screening. This adds to the repertoire of droplet assay techniques a direct protein detection method which can be performed entirely inside droplets without the need for labeling of the analyte molecules.