José Alberto Fracassi da Silva

Toner and paper‐based fabrication techniques for microfluidic applications

The interest in low‐cost microfluidic platforms as well as emerging microfabrication techniques has increased considerably over the last years. Toner‐ and paper‐based techniques have appeared as two of the most promising platforms for the production of disposable devices for on‐chip applications. This review focuses on recent advances in the fabrication techniques and in the analytical/bioanalytical applications of toner and paper‐based devices. The discussion is divided in two parts dealing with (i) toner and (ii) paper devices. Examples of miniaturized devices fabricated by using direct‐printing or toner transfer masking in polyester‐toner, glass, PDMS as well as conductive platforms as recordable compact disks and printed circuit board are presented. The construction and the use of paper‐based devices for off‐site diagnosis and bioassays are also described to cover this emerging platform for low‐cost diagnostics.

Capacitively coupled contactless conductivity detection on microfluidic systems—ten years of development

The use of capacitively coupled contactless conductivity detection (C4D) on miniaturized systems has increased considerably over the last few years. Since the first report, 10 years ago, several advances on the detection cell geometry, strategies for increasing the sensitivity and a wide range of applications have been reported. This review intends to cover the main features related to the instrumental setup of this detection method for analytical and bioanalytical assays on microfluidic chips.

A toner-mediated lithographic technology for rapid prototyping of glass microchannels

A simple, fast, and inexpensive masking technology without any photolithographic step to produce glass microchannels is proposed in this work. This innovative process is based on the use of toner layers as mask for wet chemical etching. The layouts were projected in graphic software and printed on wax paper using a laser printer. The toner layer was thermally transferred from the paper to cleaned glass surfaces (microscope slides) at 130 degrees C for 2 min. After thermal transference, the glass channel was etched using 25% (v/v) hydrofluoric acid (HF) solution. The toner mask was then removed by cotton soaked in acetonitrile. The etching rate was approximately 7.1 +/- 0.6 microm min(-1). This process is economically more attractive than conventional methods because it does not require any sophisticated instrumentation and it can be implemented in any chemical/biochemical laboratory. The glass channel was thermally bonded against a flat glass cover and its analytical feasibility was investigated using capacitively coupled contactless conductivity detection (C(4)D) and laser-induced fluorescence (LIF) detection.

Fabrication and integration of planar electrodes for contactless conductivity detection on polyester‐toner electrophoresis microchips

In this report, we describe the microfabrication and integration of planar electrodes for contactless conductivity detection on polyester‐toner (PT) electrophoresis microchips using toner masks. Planar electrodes were fabricated by three simple steps: (i) drawing and laser‐printing the electrode geometry on polyester films, (ii) sputtering deposition onto substrates, and (iii) removal of toner layer by a lift‐off process. The polyester film with anchored electrodes was integrated to PT electrophoresis microchannels by lamination at 120°C in less than 1 min. The electrodes were designed in an antiparallel configuration with 750 μm width and 750 μm gap between them. The best results were recorded with a frequency of 400 kHz and 10 Vpp using a sinusoidal wave. The analytical performance of the proposed microchip was evaluated by electrophoretic separation of potassium, sodium and lithium in 150 µm wide×6 µm deep microchannels. Under an electric field of 250 V/cm the analytes were successfully separated in less than 90 s with efficiencies ranging from 7000 to 13 000 plates. The detection limits (S/N = 3) found for K+, Na+, and Li+ were 3.1, 4.3, and 7.2 μmol/L, respectively. Besides the low‐cost and instrumental simplicity, the integrated PT chip eliminates the problem of manual alignment and gluing of the electrodes, permitting more robustness and better reproducibility, therefore, more suitable for mass production of electrophoresis microchips.

Environmental formaldehyde analysis by active diffusive sampling with a bundle of polypropylene porous capillaries followed by capillary zone electrophoretic separation and contactless conductivity detection

Compared to other volatile carbonylic compounds present in outdoor air, formaldehyde (CH(2)O) is the most toxic, deserving more attention in terms of indoor and outdoor air quality legislation and control. The analytical determination of CH(2)O in air still presents challenges due to the low-level concentration (in the sub-ppb range) and its variation with sampling site and time. Of the many available analytical methods for carbonylic compounds, the most widespread one is the time consuming collection in cartridges impregnated with 2,4-dinitrophenylhydrazine followed by the analysis of the formed hydrazones by HPLC. The present work proposes the use of polypropylene hollow porous capillary fibers to achieve efficient CH(2)O collection. The Oxyphan fiber (designed for blood oxygenation) was chosen for this purpose because it presents good mechanical resistance, high density of very fine pores and high ratio of collection area to volume of the acceptor fluid in the tube, all favorable for the development of air sampling apparatus. The collector device consists of a Teflon pipe inside of which a bundle of polypropylene microporous capillary membranes was introduced. While the acceptor passes at a low flow rate through the capillaries, the sampled air circulates around the fibers, impelled by a low flow membrane pump (of the type used for aquariums ventilation). The coupling of this sampling technique with the selective and quantitative determination of CH(2)O, in the form of hydroxymethanesulfonate (HMS) after derivatization with HSO(3)(-), by capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C(4)D) enabled the development of a complete analytical protocol for the CH(2)O evaluation in air.

Production of Calcium Oxalate Crystals by the Basidiomycete Moniliophthora perniciosa, the Causal Agent of Witches’ Broom Disease of Cacao

Oxalic acid has been shown as a virulence factor for some phytopathogenic fungi, removing calcium from pectin and favoring plant cell wall degradation. Recently, it was published that calcium oxalate accumulates in infected cacao tissues during the progression of Witches’ Broom disease (WBD). In the present work we report that the hemibiotrophic basidiomycete Moniliophthora perniciosa, the causal agent of WBD, produces calcium oxalate crystals. These crystals were initially observed by polarized light microscopy of hyphae growing on a glass slide, apparently being secreted from the cells. The analysis was refined by Scanning electron microscopy and the compositon of the crystals was confirmed by energy-dispersive x-ray spectrometry. The production of oxalate by M. perniciosa was reinforced by the identification of a putative gene coding for oxaloacetate acetylhydrolase, which catalyzes the hydrolysis of oxaloacetate to oxalate and acetate. This gene was shown to be expressed in the biotrophic-like mycelia, which in planta occupy the intercellular middle-lamella space, a region filled with pectin. Taken together, our results suggest that oxalate production by M. perniciosa may play a role in the WBD pathogenesis mechanism.

Rapid prototyping of polymeric electrophoresis microchips with integrated copper electrodes for contactless conductivity detection

A simple and easy approach to produce polymeric microchips with integrated copper electrodes for capacitively coupled contactless conductivity detection (C4D) is described. Copper electrodes were fabricated using a printed circuit board (PCB) as an inexpensive thin-layer of metal. The electrode layout was first drawn and laser printed on a wax paper sheet. The toner layer deposited on the paper sheet was thermally transferred to the PCB surface working as a mask for wet chemical etching of the copper layer. After the etching step, the toner was removed with an acetonitrile-dampened cotton. A poly(ethylene terephthalate) (PET) film coated with a thin thermo-sensitive adhesive layer was used to laminate the PCB plate providing an insulator layer of the electrodes to perform C4D measurements. Electrophoresis microchannels were fabricated in poly(dimethylsiloxane) (PDMS) by soft lithography and reversibly sealed against the PET film. These hybrid PDMS/PET chips exhibited a stable electroosmotic mobility of 4.25 ± 0.04 × 10-4 V cm-2 s-1, at pH 6.1, over fifty runs. Efficiencies ranging from 1127 to 1690 theoretical plates were obtained for inorganic cations.

Monitoring intracellular nitric oxide production using microchip electrophoresis and laser-induced fluorescence detection

Nitric oxide (NO) is a biologically important short-lived reactive species that has been shown to be involved in a large number of physiological processes. The production of NO is substantially increased in immune and other cell types through the upregulation of inducible nitric oxide synthase (iNOS) caused by exposure to stimulating agents such as lipopolysaccharide (LPS). NO production in cells is most frequently measured via fluorescence microscopy using diaminofluorescein-based probes. Capillary electrophoresis with laser-induced fluorescence detection has been used previously to separate and quantitate the fluorescence derivatives of NO from potential interferences in single neurons. In this paper, microchip electrophoresis (ME) coupled to laser-induced fluorescence (LIF) detection is evaluated as a method for measurement of the NO production by Jurkat cells under control and stimulating conditions. ME is ideal for such analyses due to its fast and efficient separations, low volume requirements, and ultimate compatibility with single cell chemical cytometry systems. In these studies, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA) was employed for the detection of NO, and 6-carboxyfluorescein diacetate (6-CFDA) was employed as an internal standard. Jurkat cells were stimulated using lipopolysaccharide (LPS) to produce NO, and bulk cell analysis was accomplished using ME-LIF. Stimulated cells exhibited an approximately 2.5-fold increase in intracellular NO production compared to the native cells. A NO standard prepared using diethylamine NONOate (DEA/NO) salt was used to construct a calibration curve for quantitation of NO in cell lysate. Using this calibration curve, the average intracellular NO concentrations for LPS-stimulated and native Jurkat cells were calculated to be 1.5 mM and 0.6 mM, respectively

Simultaneous determination of free fluoride and monofluorophosphate in toothpaste by capillary electrophoresis with capacitively coupled contactless conductivity detection.

Capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C(4)D) was used for rapid, accurate and simultaneous determination of free fluoride and monofluorophosphate (MFP) in six different toothpaste samples. A buffer solution containing 15 mmol L(-1) histine, 25 mmol L(-1) lactic acid, and 2.5 mmol L(-1) tetradecyltrimethylammonium bromide (TTAB) was used as background electrolyte (BGE). A complete separation of the analytes and the internal standard (tartrate) could be attained in less than 2.5 min. The limits of detection (LOD) and quantification (LOQ) were, respectively, 0.17 and 0.57 mg L(-1) for free fluoride and 0.70 and 2.33 mg L(-1) for MFP. Recoveries ranging from 85 to 107% were obtained for samples spiked with standard solutions of free fluoride or MFP. The CE-C(4)D method was compared to an ion-selective electrode (ISE) method and the results were in good agreement. More importantly, the CE-C(4)D method demonstrates the advantage of being able to determine MFP without a prior hydrolysis step.

Use of experimental design and effective mobility calculations to develop a method for the determination of antimicrobials by capillary electrophoresis

A capillary zone electrophoresis (CZE) method for the determination of chloramphenicol (CLP), danofloxacin (DANO), ciprofloxacin (CIPRO), enrofloxacin (ENRO), sulfamethazine (SMZ), sulfaquinoxaline (SQX) and sulfamethoxazole (SMX) is described. For the development, the effective mobilities were estimated and a central composite design was performed. The method was in-house validated for CLP, CIPRO, ENRO and SMX determination in pharmaceuticals. In comparison with the HPLC method recommended by the United States Pharmacopoeia, this CZE method exhibited the same performance, with the advantage that seven different antimicrobials in pharmaceutical formulations could be simultaneously determined.