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Dr Lucas Blanes

Biography

Dr Lucas Blanes is Biologist (2001), MSc in Biochemistry (2004) and PhD in Analytical Chemistry (2008) from Sao Paulo University – Brazil.

His master degree was focused in the purification and characterization of enzymes and his PhD in the development of capillary electrophoresis instruments and sensors to analyze biomolecules. He did part of his Ph.D at University of Texas at San Antonio (EUA) developing Lab-on-a-chip devices.

Since 2008 he is a postdoc at UTS. His research is focused in the development of portable microfluidic devices and analysis of Forensic samples.

Image of Lucas Blanes
Postdoctoral Fellow, School of Mathematical and Physical Sciences
B Biological Sciences, M Sc, Doctor of Chemistry
 

Research Interests

  • Chemical analysis of forensic, environmental and biochemical species.
  • Capillary (CE) and Microchip capillary electrophoresis - Lab on a chip (opens an external site).
  • Development of paper-based microchips (MicroPads) for low-cost diagnostics for all (opens an external site).
  • Design and build analytical instruments and sensors
  • Chip Liquid chromatography-Inductively coupled plasma-mass spectrometry for metallomics and cancer analysis

Can supervise: Yes

  • Capillary electrophoresis
  • Microchip Electrophoresis (Lab-on-a-chip)
  • Paper microfluidics
  • Analytical chemistry
  • Forensic, chemical and biochemical analysis.
  • Development of Portable analytical instruments

Chapters

do Lago, C.L., Nogueira, T., Blanes, L. & Saito, R.M. 2013, 'Determination of mono-, di-, and oligosaccharides by capillary electrophoresis with capacitively coupled contactless conductivity detection' in Volpi, N. & Maccari, F.E. (eds), Methods in Molecular Biology, Humana Press, Springer Science, pp. 51-60.
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Saccharides and chitooligosaccharides can be separated in electrophoretic conditions by raising the pH of the medium, which renders the corresponding alcoholate forms. These anionic species can be separated and detected with capacitively coupled contactless conductivity detection as negative peaks because of their low mobilities when compared to the hydroxyl mobility, which is the main co-ion in the background electrolyte. Three methods for different matrixes are presented in this chapter.
Blanes, L., Tomazelli Coltro, W.K., Saito, R.M., do Lago, C.L., Roux, C.P. & Doble, P.A. 2013, 'Practical considerations for the design and implementation of High Voltage Power Supplies for Capillary and Microchip Capillary Electrophoresis' in Carlos, D.G.A., Karin, Y.C.T. & Emanuel, C. (eds), Capillary Electrophoresis and Microchip Capillary Electrophoresis: Principles, Applications, and Lim, Wiley, Chichester, pp. 67-76.
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Capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE) are powerful analytical techniques used to analyze chemical and biological samples. For both techniques sample injection and separation are two crucial steps that depend on a reliable high-voltage power supply (HVPS) to ensure reproducible separations. Therefore, the source of high voltage (HV) is considered to be the heart for these instruments. Separation of the analytes occurs due to the influence of an applied potential difference between electrodes placed at the ends of the capillary or channel. As a consequence, the components present in the plug of injection are driven toward the detector. This book chapter is a comprehensive source of information of HVPS for CE and MCE. This chapter covers topics as such as fundamentals of HV, electroosmotic flow control, construction of bipolar HVPS from unipolar HVPS, commercially available HVPS and DC/DC converters, alternative sources of HV, HVPS controllers for MCE, and strategies to measure HV. The chapter also includes practical and safety considerations that can be helpful for development of new CE and MCE instrumentation
Van Gramberg, A.A., Beavis, A.B., Blanes, L. & Doble, P.A. 2010, 'Optimisation Of The Separation Of Amino Acids By Capillary Electrophoresis Using Artificial Neural Networks' in Hanrahan, G. & Gomez, F.A. (eds), Chemometric Methods in Capillary Electrophoresis, John Wiley & Sons, Inc, United States, pp. 169-180.
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Many factors can affect the separation performance of a capillary electrophoresis (CE) electrolyte, such as the buffer, surfactant and organic modifier concentrations, pH, capillary temperature, and applied voltage (1). The efficient manipulation of these factors is critical to optimize the resolution of a given analysis in the shortest time frame. During the method development process, an analyst will usually attempt a separation based on a previously reported method that is similar or the same as the requirements of the analysis at hand. If the separation is inadequate, a univariate approach (2) is often employed to attempt to improve the separation. This involves altering one parameter at a time in a systematic way, and viewing the results by plotting the effect of the parameter on the migration time of the analytes. In this way, suitable electrolyte compositions may be found that separates all of the analytes. If suitable conditions are not found, a second electrolyte parameter is chosen and altered in a similar manner. This univariate procedure is then repeated until a suitable condition is found. This method of optimization is time-consuming, and it is unknown if the optimum is truly the global optimum.
Funes-Huacca, M.E., Alberice, J.V., Blanes, L. & Carrilho, E. 2010, 'Chemometric Methods Applied to Genetic Analyses by Capillary Electrophoresis and Electrophoresis Microchip Technologies' in Grady Hanrahan (ed), Chemometric Methods in Capillary Electrophoresis, John Wiley & Sons, Inc, United States, pp. 261-290.
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In this chapter we have summarized the complex issues that are involved in the analysis of sizing DNA by capillary electrophoresis (CE), and how chemometric methods can help to optimize a high number of interrelated variables. It is impressive to observe how diverse is the biological information obtainable despite the size of the double-stranded DNA molecule. We also briefly introduce some typical genetic assays that rely on sizing DNA molecules, and how some chemometric approaches are used to correlate sizes of DNA with population and or evolution of species.

Conferences

Blanes, L., Taudte, R.V., Roux, C. & Doble, P. 2014, 'Using paper-based microfluidics and lab on a chip techonologies for the rapid analysis of trinitro aromatic explosives', 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, pp. 1581-1582.
&copy; 14CBMS. We report a new microfluidic paper-based analytical device (PAD) containing potassium hydroxide to create a color change reaction when in contact with trinitro aromatic explosives, followed by extraction and lab on a chip analysis. The procedure allowed the detection and identification of 1 g of the target explosives distributed on a 100 cm<sup>2</sup>surface.

Journal articles

Ueland, M., Blanes, L., Taudte, R.V., Stuart, B.H., Cole, N., Willis, P., Roux, C. & Doble, P. 2016, 'Capillary-driven microfluidic paper-based analytical devices for lab on a chip screening of explosive residues in soil.', Journal of chromatography. A, vol. 1436, pp. 28-33.
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A novel microfluidic paper-based analytical device (PAD) was designed to filter, extract, and pre-concentrate explosives from soil for direct analysis by a lab on a chip (LOC) device. The explosives were extracted via immersion of wax-printed PADs directly into methanol soil suspensions for 10min, whereby dissolved explosives travelled upwards into the PAD circular sampling reservoir. A chad was punched from the sampling reservoir and inserted into a LOC well containing the separation buffer for direct analysis, avoiding any further extraction step. Eight target explosives were separated and identified by fluorescence quenching. The minimum detectable amounts for all eight explosives were between 1.4 and 5.6ng with recoveries ranging from 53-82% from the paper chad, and 12-40% from soil. This method provides a robust and simple extraction method for rapid identification of explosives in complex soil samples.
Taudte, R.V., Roux, C., Blanes, L., Horder, M., Kirkbride, K.P. & Beavis, A. 2016, 'The development and comparison of collection techniques for inorganic and organic gunshot residues', ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 408, no. 10, pp. 2567-2576.
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Evans, E., Costrino, C., do Lago, C.L., Garcia, C.D., Roux, C. & Blanes, L. 2016, 'Determination of Inorganic Ion Profiles of Illicit Drugs by Capillary Electrophoresis', Journal of Forensic Sciences.
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&copy; 2016 American Academy of Forensic Sciences.A portable capillary electrophoresis instrument with dual capacitively coupled contactless conductivity detection (C4D) was used to determine the inorganic ionic profiles of three pharmaceutical samples and precursors of two illicit drugs (contemporary samples of methylone and para-methoxymethamphetamine). The LODs ranged from 0.10 mol/L to 1.25 mol/L for the 10 selected cations, and from 0.13 mol/L to 1.03 mol/L for the eight selected anions. All separations were performed in less than 6 min with migration times and peak area RSD values ranging from 2 to 7%. The results demonstrate the potential of the analysis of inorganic ionic species to aid in the identification and/or differentiation of unknown tablets, and real samples found in illicit drug manufacture scenarios. From the resulting ionic fingerprint, the unknown tablets and samples can be further classified.
Morelato, M., Barash, M., Blanes, L., Chadwick, S., Dilag, J., Kuzhiumparambil, U., Nizio, K.D., Spindler, X. & Moret, S. 2016, 'Forensic Science: Current State and Perspective by a Group of Early Career Researchers', Foundations of Science, pp. 1-27.
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Forensic science and its influence on policing and the criminal justice system have increased since the beginning of the twentieth century. While the philosophies of the forensic science pioneers remain the pillar of modern practice, rapid advances in technology and the underpinning sciences have seen an explosion in the number of disciplines and tools. Consequently, the way in which we exploit and interpret the remnant of criminal activity are adapting to this changing environment. In order to best exploit the trace, an interdisciplinary approach to both research and investigation is required. In this paper, nine postdoctoral research fellows from a multidisciplinary team discuss their vision for the future of forensic science at the crime scene, in the laboratory and beyond. This paper does not pretend to be exhaustive of all fields of forensic science, but describes a portion of the postdoctoral fellows' interests and skills.
Moraes, M.L.L., da Silva, H.D.T., Blanes, L., Dobb, P. & Tavares, M.F.M. 2016, 'Optimization of chemometric approaches for the extraction of isorhamnetin-3-O-rutinoside from Calendula officinalis L.', Journal of Pharmaceutical and Biomedical Analysis, vol. 125, pp. 408-414.
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&copy; 2016.The application of Design of Experiments (DoE) to the determination of optimum conditions for an extraction process relies on the correct selection of mathematical models. The linear model is the one typically used; however, in some cases it does not always have superior performance, ignoring the real nature of the data and its appropriate descriptive model. In order to evaluate the extraction efficiency of isorhamnetin-3-O-rutinoside from flowers of Calendula officinalis L. a multivariate factorial analysis was used. Simulations were conducted using linear, quadratic, full cubic and special cubic models. A Simplex-Centroid design was chosen as it delivered greater precision with only minor errors versus other models tested. Analyses were performed by capillary zone electrophoresis using sodium tetraborate buffer (40 mmol L-1, pH 9.4) containing 10% methanol. The detection was linear over a range of 8.0-50.0 mg L-1 (r2 = 0.996), and the limits of detection (LOD) and quantification (LOQ) for isorhamnetin-3-O-rutinoside were 3.44 mg L-1 and 11.47 mg L-1, respectively. The full cubic model showed the best extraction results, with an error of 3.40% compared to analysis of variance, and a determination coefficient of 0.974. The difference between the responses at the reference point, calculated by the model, and the experimental response, varies around 2.72% for full cubic model. Comparison of the four models showed the full cubic model was the most appropriate one, allowing greater efficiency in the extraction of isorhamnetin-3-O-rutinoside. Selection of the model made it possible to obtain a 60% increase in sensitivity compared to the linear model.
Taudte, R.V., Roux, C., Bishop, D., Blanes, L., Doble, P. & Beavis, A. 2015, 'Development of a UHPLC method for the detection of organic gunshot residues using artificial neural networks', Analytical Methods, vol. 7, no. 18, pp. 7447-7454.
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The introduction of lead and heavy-metal free ammunition to the market challenges the current protocol for gunshot residue (GSR) investigations, which focuses on the inorganic components. Future proofing GSR analysis requires the development and implementation of new methods for the collection and analysis of organic GSR (OGSR) into operational protocols. This paper describes the development and optimisation of an ultra high performance liquid chromatography method for the analysis of 32 compounds potentially present in OGSR. An artificial neural network was applied to predict the retention times of the target analytes for various gradients for rapid determination of optimum separation conditions. The final separation and analysis time for the 32 target analytes was 27 minutes with limits of detection ranging from 0.03 to 0.21 ng. The method was applied to the analysis of smokeless powder and samples collected from the hands of a shooter following the discharge of a firearm. The results demonstrate that the method has the potential for use in cases involving GSR.
Peters, K.L., Corbin, I., Kaufman, L.M., Zreibe, K., Blanes, L. & McCord, B.R. 2015, 'Simultaneous colorimetric detection of improvised explosive compounds using microfluidic paper-based analytical devices (PADs)', Analytical Methods, vol. 7, no. 1, pp. 63-70.
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&copy; 2014 The Royal Society of Chemistry.In this paper the development of microfluidic paper-based analytical devices (PADs) is described for rapid, on-site detection of improvised explosives. Five lane PADs were designed and printed using wax ink on chromatography paper to create hydrophobic channels. Each channel contains colorimetric reagents capable of reacting with one or more explosive compounds resulting in a specific colorimetric reaction. Two devices were prepared, each capable of performing five simultaneous analyses on a single PAD. The first PAD was developed to detect inorganic explosives such as black powder, flash powder, and ammonium nitrate. It detects nitrate, nitrite, chlorate, and perchlorate oxidizers, as well as ammonium. The second PAD was developed to detect military explosives such as TNT and RDX along with other high explosives like urea nitrate. It also detects organic peroxides such as TATP and its precursor hydrogen peroxide. All experiments were performed by dissolving the explosives in deionized water or 50 : 50 acetone/H2O as transport solvents with a detection time of around 5 minutes. Detection limits ranged from 0.39-19.8 g of explosive compound. These two customized PAD devices permit the on-site forensic testing of unknown explosives, thereby supplying law enforcement and military personnel with a resource for fast, easy detection of military, commercial, and homemade explosive components at low cost.
Lloyd, A.E., Russell, M., Blanes, L., Somerville, R., Doble, P.A. & Roux, C.P. 2014, 'The application of portable microchip electrophoresis for the screening and comparative analysis of synthetic cathinone seizures', Forensic Science International, vol. 242, pp. 16-23.
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Variation in the chemical composition of illicit tablets and powders is common among samples within a given drug seizure. Using microchip electrophoresis (ME), multiple tablets can be screened in a cost-effective and timely manner. This method could be used in conjunction with reporting methods that focus solely on statistical sampling to infer homogeneity or otherwise of a larger subset of tablets. Some frequently observed synthetic cathinones, often present in illicit tablets seized in New Zealand, were chosen for analysis. An ME device (Agilent Bioanalyzer 2100) was used to electrophoretically separate synthetic cathinones. The background electrolyte was composed of a 50mM sodium tetraborate buffer with 50mM sodium dodecyl sulphate at pH 9.66. Analytes were derivatised prior to analysis for 3min at 90&deg;C, employing fluorescein isothiocyanate isomer I (FITC). A characteristic fluorescent profile was obtained for each tablet, in terms of the number of constituents, relative peak height ratios and migration times. The repeatability of the developed method was assessed for a wide range of tablets and relative standard deviations of 0.4-5.2% and 1.6-5.5% were calculated for migration times and peak height ratios, respectively. The use of microchip tablet profiles in the forensic case comparison of illicit drug seizure samples in realistic scenarios is discussed.
Pesenti, A., Taudte, R.V., McCord, B., Doble, P.A., Blanes, L. & Roux, C.P. 2014, 'Coupling Paper-Based Microfluidics and Lab on a Chip Technologies for Confirmatory Analysis of Trinitro Aromatic Explosives', Analytical Chemistry, vol. 86, pp. 4707-4714.
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A new microfluidic paper-based analytical device (&igrave;PAD) in conjunction with confirmation by a lab on chip analysis was developed for detection of three trinitro aromatic explosives. Potassium hydroxide was deposited on the &igrave;PADs (0.5 &igrave;L, 1.5 M), creating a color change reaction when explosives are present, with detection limits of approximately 7.5 &plusmn; 1.0 ng for TNB, 12.5 &plusmn; 2.0 ng for TNT and 15.0 &plusmn; 2.0 ng for tetryl. For confirmatory analysis, positive &igrave;PADs were sampled using a 5 mm hole-punch, followed by extraction of explosives from the punched chad in 30 s using 20 &igrave;L borate/SDS buffer. The extractions had efficiencies of 96.5 &plusmn; 1.7%. The extracted explosives were then analyzed with the Agilent 2100 Bioanalyzer lab on a chip device with minimum detectable amounts of 3.8 &plusmn; 0.1 ng for TNB, 7.0 &plusmn; 0.9 ng for TNT, and 4.7 &plusmn; 0.2 ng for tetryl. A simulated in-field scenario demonstrated the feasibility of coupling the &igrave;PAD technique with the lab on a chip device to detect and identify 1 &igrave;g of explosives distributed on a surface of 100 cm2.
Taudte, R.V., Beavis, A.B., Blanes, L., Cole, N.A., Doble, P.A. & Roux, C.P. 2014, 'Detection of Gunshot Residues Using Mass Spectrometry', BioMed Research International, vol. 965403, pp. 1-16.
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In recent years, forensic scientists have become increasingly interested in the detection and interpretation of organic gunshot residues (OGSR) due to the increasing use of lead- and heavy metal-free ammunition. This has also been prompted by the identification of gunshot residue- (GSR-) like particles in environmental and occupational samples. Various techniques have been investigated for their ability to detect OGSR.Mass spectrometry (MS) coupled to a chromatographic system is a powerful tool due to its high selectivity and sensitivity. Further,modernMS instruments can detect and identify a number of explosives and additives whichmay require different ionization techniques. Finally,MS has been applied to the analysis of bothOGSR and inorganic gunshot residue (IGSR), although the gold standard for analysis is scanning electron microscopy with energy dispersive X-ray microscopy (SEM-EDX). This review presents an overview of the technical attributes of currently available MS and ionization techniques and their reported applications to GSR analysis.
Porto, S.K.S.S., Nogueira, T., Blanes, L., Doble, P., Sabino, B.D., do Lago, C.L. & Angnes, L. 2014, 'Analysis of ecstasy tablets using capillary electrophoresis with capacitively coupled contactless conductivity detection.', Journal of forensic sciences, vol. 59, no. 6, pp. 1622-1626.
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A method for the identification of 3,4-methylenedioxymethamphetamine (MDMA) and meta-chlorophenylpiperazine (mCPP) was developed employing capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C(4) D). Sample extraction, separation, and detection of "Ecstasy" tablets were performed in <10&nbsp;min without sample derivatization. The separation electrolyte was 20&nbsp;mm TAPS/Lithium, pH 8.7. Average minimal detectable amounts for MDMA and mCPP were 0.04&nbsp;mg/tablet, several orders of magnitude lower than the minimum amount encountered in a tablet. Seven different Ecstasy tablets seized in Rio de Janeiro, Brazil, were analyzed by CE-C(4) D and compared against routine gas chromatography-mass spectrometry (GC-MS). The CE method demonstrated sufficient selectivity to discriminate the two target drugs, MDMA and mCPP, from the other drugs present in seizures, namely amphepramone, fenproporex, caffeine, lidocaine, and cocaine. Separation was performed in <90&nbsp;sec. The advantages of using C(4) D instead of traditional CE-UV methods for in-field analysis are also discussed.
Taudte, R.V., Beavis, A.B., Wilson-Wilde, L., Roux, C.P., Doble, P.A. & Blanes, L. 2013, 'A portable explosive detector based on fluorescence quenching of pyrene deposited on coloured wax-printed µpADs', Lab on a Chip - Miniaturisation for Chemistry,Physics, Biology, materials science and bioengineering, vol. 13, no. 21, pp. 4164-4172.
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A new technique for the detection of explosives has been developed based on fluorescence quenching of pyrene on paper-based analytical devices (&micro;PADs). Wax barriers were generated (150 &deg;C, 5 min) using ten different colours. Magenta was found as the most suitable wax colour for the generation of the hydrophobic barriers with a nominal width of 120 &micro;m resulting in fully functioning hydrophobic barriers. One microliter of 0.5 mg mL-1 pyrene dissolved in an 80:20 methanolwater solution was deposited on the hydrophobic circle (5 mm diameter) to produce the active microchip device. Under ultra-violet (UV) illumination, ten different organic explosives were detected using the &micro;PAD, with limits of detection ranging from 100600 ppm. A prototype of a portable battery operated instrument using a 3 W power UV light-emitting-diode (LED) (365 nm) and a photodiode sensor was also built and evaluated for the successful automatic detection of explosives and potential application for field-based screening.
Lloyd, A.E., Russell, M., Blanes, L., Doble, P.A. & Roux, C.P. 2013, 'Lab-on-a-chip screening of methamphetamine and pseudoephedrine in samples from clandestine laboratories', Forensic Science International, vol. 228, no. 1-3, pp. 8-14.
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The clandestine manufacture of methamphetamine in New Zealand predominantly involves the reduction of pseudoephedrine, extracted from pharmaceutical preparations, using hydrogen iodide. This method of illicit manufacture leaves a variety of materials at the scene that are a rich source of information. Efficient processing and preliminary identification of extraction and reaction mixtures, precursors and products is essential to minimise exposure to potential hazardous materials and to provide investigative and intelligence information. In this study, we employed a portable lab-on-a-chip instrument for the rapid and cost effective screening of methamphetamine, pseudoephedrine and ephedrine in a variety of sample types found in typical clandestine laboratory scenarios
Blanes, L., Tomazelli Coltro, W.K., Saito, R.M., Van Gramberg, A.A., do Lago, C.L. & Doble, P.A. 2012, 'High-voltage power supplies to capillary and microchip electrophoresis', Electrophoresis, vol. 33, no. 6, pp. 893-898.
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Over the past years, the development of capillary electrophoresis (CE) and microchip electrophoresis (ME) systems has grown due to instrumental simplicity and wide application. In both CE andME, the application of a high voltage (HV) is a crucial step in the electrokinetic (EK) injection and separation processes. Particularly on ME devices, EK injection is often performed with three different modes: gated, pinched, and unpinched. In all these cases, different potential values may be applied to one or multiple channels to control the injection of small sample volumes as well as the separation process. For this reason, the construction of reliable HV power supplies (HVPS) is required. This review covers the advances of the development of commercial and laboratory-built HVPS for CE and ME. Moreover, it intends to be a guide for new developers of electrophoresis instrumentation.
Lloyd, A.E., Blanes, L., Beavis, A.B., Roux, C.P. & Doble, P.A. 2011, 'A Rapid Method For The In-Field Analysis Of Amphetamines Employing The Agilent Bioanalyzer', Analytical Methods, vol. 3, no. 7, pp. 1535-1539.
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This paper reports the first analysis of small molecules on the Agilent bio-analyser. The Bioanalyzer is a commercial lab-on-a-chip instrument designed for the analysis of DNA and proteins. We demonstrate that the instrument is suitable for analyses beyo
Saito, R.M., Brito-Neto, J.G., Lopes, F.S., Blanes, L., Costa, E.T., Vidal, D.T., Hotta, G.M. & do Lago, C.L. 2010, 'Ionic mobility of the solvated proton and acid-base titration in a fourcompartment capillary electrophoresis system', Analytical Methods, vol. 2, no. 2, pp. 164-170.
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Although H+ and OH are the most common ions in aqueous media, they are not usually observable in capillary electrophoresis (CE) experiments, because of the extensive use of buffer solutions as the background electrolyte. In the present work, we introduce CE equipment designed to allow the determination of such ions in a similar fashion as any other ion. Basically, it consists of a fourcompartment piece of equipment for electrolysis-separated experiments (D. P. de Jesus et al., Anal. Chem., 2005, 77, 607). In such a system, the ends of the capillary are placed in two reservoirs, which are connected to two other reservoirs through electrolyte-filled tubes. The electrodes of the high-voltage power source are positioned in these reservoirs. Thus, the electrolysis products are kept away from the inputs of the capillary. The detection was provided by two capacitively coupled contactless conductivity detectors (C4D), each one positioned about 11 cm from the end of the capillary. Two applications were demonstrated: titration-like procedures for nanolitre samples and mobility measurements. Strong and weak acids (pKa < 5), pure or mixtures, could be titrated. The analytical curve is linear from 50 mMup to 10 mM of total dissociable hydrogen (r &frac14; 0.99899 for n &frac14; 10) in 10-nL samples. By including D2O in the running electrolyte, we could demonstrate how to measure the mixed proton/deuteron mobility. When H2O/D2O (9 : 1 v/v) was used as the solvent, the mobility was 289.6 0.5 105 cm2 V1 s1. Due to the fast conversion of the species, this value is related to the overall behaviour of all isotopologues and isotopomers of the Zundel and Eigen structures, as well as the Stokesian mobility of proton and deuteron. The effect of neutral (o-phenanthroline) and negatively charged (chloroacetate) bases and aprotic solvent (DMSO) over the H+ mobility was also demonstrated.
Felhofer, J.L., Blanes, L. & Garcia, C.D. 2010, 'Recent developments in instrumentation for capillary electrophoresis and microchip-capillary electrophoresis', Electrophoresis, vol. 31, no. 15, pp. 2469-2486.
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Over the last years, there has been an explosion in the number of developments and applications of CE and microchip-CE. In part, this growth has been the direct consequence of recent developments in instrumentation associated with CE. This review, which is focused on the contributions published in the last 5 years, is intended to complement the articles presented in this special issue dedicated to instrumentation and to provide an overview of the general trends and some of the most remarkable developments published in the areas of high-voltage power supplies, detectors, auxiliary components, and compact systems. It also includes a few examples of alternative uses of and modifications to traditional CE instruments.
Epple, R., Blanes, L., Beavis, A., Roux, C. & Doble, P. 2010, 'Analysis of amphetamine-type substances by capillary zone electrophoresis using capacitively coupled contactless conductivity detection.', Electrophoresis, vol. 31, no. 15, pp. 2608-2613.
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CE with capacitively coupled contactless conductivity detection (C(4)D) was employed for the separation and detection of seven amphetamine analogues as well as amphetamine, dextroamphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine. The separation electrolyte was 30 mM hydroxypropyl-beta-cyclodextrin (HPbetaCD) in a 75 mM acetic acid+25 mM sodium acetate buffer adjusted to pH 4.55. Conductivity detection was compared with UV detection using this same electrolyte. Average detection limits for C(4)D and UV were 1.3 and 1.0 ppm, respectively. The effects of HPbetaCD concentration and BGE composition on the selectivity of the separation were also investigated. An illicit, street-grade sample of 3,4-methylenedioxymethamphetamine (Ecstasy) and a prescription dextroamphetamine tablet were also analysed.
Blanes, L., Saito, R.M., Genta, F.A., Donega, J., Terra, W.R., Ferreira, C. & do Lago, C.L. 2008, 'Direct detection of underivatized chitooligosaccharides produced through chitinase action using capillary zone electrophoresis', Analytical Biochemistry, vol. 373, no. 1, pp. 99-103.
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Capillary electrophoresis with capacitively coupled contactless conductivity detection was successfully used to quantify N-acetylglucosamine and five N-acetyl-chitooligosaccharides (C2C6) produced after reaction with a purified chitinase (TmChi) from Tenebrio molitor (Coleoptera). No derivatization process was necessary. The separation was developed using 10 mM NaOH with 10% (v/v) acetonitrile as background electrolyte and homemade equipment with a system that avoids the harmful effect of electrolysis. The limit of detection for all oligosaccharides was ca. 3 lM, and the results indicated that the larger the oligosaccharide, the higher the sensitivity. Analysis of the chitooligosaccharides produced revealed that TmChi has an endolytic cleavage pattern with C5 as the best substrate (higher catalytic efficiency kcat/KM) releasing C2 and C3.
Saito, R.M., Neves, C.A., Lopes, F.S., Blanes, L., Brito-Neto, J.G. & do Lago, C.L. 2007, 'Monitoring the electroosmotic flow in capillary electrophoresis using contactless conductivity detection and thermal marks', Analytical Chemistry, vol. 79, no. 1, pp. 215-223.
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The fundamental aspects and the capillary electrophoresis usage of thermal marks are presented. The so-called thermal mark is a perturbation of the electrolyte concentration generated by a punctual heating of the capillary while the separation electric field is maintained. The heating pulse is obtained by powering tungsten filaments or surface mount device resistors with 5 V during a few tens to hundreds of milliseconds. In the proposed model, the variation of the transport numbers with the rising temperature leads to the formation of low- and highconcentration regions during the heating. After cooling down, the initial mobilities of the species are restored and these regions (the thermal mark) migrate chiefly due to the electroosmotic flow (EOF). The mark may be recorded with a conductivity detector as part of a usual electropherogram and be used to index the analyte peaks and thus compensate for variations of the EOF. In a favorable case, 10 mmol/L KCl solution, the theory suggests that the error in the measurement of EOF mobility by this mean is only -6.5 10-7 cm2 V-1 s-1. The method was applied to the analysis of alkaline ions in egg white, and the relative standard deviations of the corrected mobilities of these ions were smaller than 1%.
Blanes, L., Mora, M.F., do Lago, C.L., Ayon, A. & Garcia, C.D. 2007, 'Lab-on-a-chip biosensor for glucose based on a packed immobilized enzyme reactor', Electroanalysis, vol. 19, no. 23, pp. 2451-2456.
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In this work, the development of a packed immobilized enzyme reactor (IMER) and its integration to a capillary electrophoresis microchip is described. The present microchip design differs from others, in the fact that the same design could be used with or without the particles and, just by changing the material used to pack the IMER, different analytes can be detected. The applied procedure involves the separation of the target analyte by capillary electrophoresis (CE), which is then coupled to a post-column IMER that produces H2O2. The H2O2 produced is finally detected downstream at the surface of a working electrode. Glucose was detected above 100 mM by packing particles modified with glucose oxidase at the end of the separation channel. The analytical performance of the microchip-CE has been demonstrated by performing the separation and detection of glucose and noradrenaline. Additions of fructose showed no effect on either the peak position or the peak magnitude of glucose. The microchip-CE-IMER was also used to quantify glucose in carbonated beverages with good agreement with other reports.
de Jesus, D.P., Blanes, L. & do Lago, C.L. 2006, 'Microchip free-flow electrophoresis on glass substrate using laser-printing toner as structural material', Electrophoresis, vol. 27, no. 24, pp. 4935-4942.
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In this work, a microfluidic free flow electrophoresis device, obtained by thermal toner transferring on glass substrate, is presented. A microdevice can be manufactured in only 1 h. The layout of the microdevice was designed in order to improve the fluidic and electrical characteristics. The separation channel is 8 mm deep and presents an internal volume of 1.42 mL. The deleterious electrolysis effects were overcome by using a system that isolates the electrolysis products from the separation channel. The Joule heating dissipation in the separation channel was found to be very efficient up to a current density of 8.83mA/mm2 that corresponds to a power dissipation per unit volume of running electrolyte of 172mW/mL. Promising results were obtained in the evaluation of the microdevices for the separation of ionic dyes. The microfluidic device can be used for a continuous sample pretreatment step for micro total analysis system.
Genta, F.A., Blanes, L., Cristofoletti, P.T., do Lago, C.L., Terra, W.R. & Ferreira, C. 2006, 'Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut', Insect Biochemistry and Molecular Biology, vol. 36, pp. 789-800.
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Insect chitinases are involved in degradation of chitin from the exoskeleton cuticle or from midgut peritrophic membrane during molts. cDNAs coding for insect cuticular and gut chitinases were cloned, but only chitinases from moulting fluid were purified and characterized. In this study the major digestive chitinase from T. molitor midgut (TmChi) was purified to homogeneity, characterized and sequenced after cDNA cloning. TmChi is secreted by midgut epithelial cells, has a molecular weight of 44 kDa and is unstable in the presence of midgut proteinases. TmChi shows strong substrate inhibition when acting on umbelliferyl-derivatives of chitobio- and chitotriosaccharides, but has normal Michaelis kinetics with the N-acetylglucosamine derivative as substrate. TmChi has very low activity against colloidal chitin, but effectively converts oligosaccharides to shorter fragments. The best substrate for TmChi is chitopentaose, with highest kcat/KM value. Sequence analysis and chemical modification experiments showed that the TmChi active site contains carboxylic groups and a tryptophane, which are known to be important for catalysis in family 18 chitinases. Modification with p-hidroximercuribenzoate of a cysteine residue, which is exposed after substrate binding, leads to complete inactivation of the enzyme. TmChi mRNA encodes a signal peptide plus a protein with 37 kDa and high similarity with other insect chitinases from family 18. Surprisingly, this gene does not encode the C-terminal SerThr-rich connector and chitin-binding domain normally present in chitinases. The special features of TmChi probably result from its adaptation to digest chitin-rich food without damaging the peritrophic membrane.
Brito-Neto, J.G., Silva, J.A., Blanes, L. & do Lago, C.L. 2005, 'Understanding capacitively coupled contactless conductivity detection in capillary and microchip electrophoresis. Part 1. Fundamentals', Electroanalysis, vol. 17, no. 13, pp. 1198-1206.
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Capacitively coupled contactless conductivity detection (C4D) is presented in a progressively detailed approach. Through different levels of theoretical and practical complexity, several aspects related to this kind of detection are addressed, which should be helpful to understand the results as well as to design a detector or plan experiments. Simulations and experimental results suggest that sensitivity depends on: 1) the electrolyte co-ion and counter-ion; 2) cell geometry and its positioning; 3) operating frequency. Undesirable stray capacitance formed due to the close placement of the electrodes is of great importance to the optimization of the operating frequency and must be minimized.
Brito-Neto, J.G., Silva, J.A., Blanes, L. & do Lago, C.L. 2005, 'Understanding capacitively coupled contactless conductivity detection in capillary and microchip electrophoresis. Part 2. Peak shape, stray capacitance, noise, and actual electronics', Electroanalysis, vol. 17, no. 13, pp. 1207-1214.
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Although simple equivalent circuits have been used to explain the basic functioning of a capacitively coupled contactless conductivity detector (C4D), more sophisticated models are required to take into account the effects of the spatial non-homogeneity of the solution conductivity as the electrophoretic zones pass inside the detector. The overshooting phenomenon observed in real electropherograms may be explained by modeling the coupling of the electrodes with the inner capillary with a network of resistors and capacitors and its dependence with the stray capacitance becomes evident. An even more detailed model of the cell based on electrostatics allows one to calculate the stray capacitances. For the typical geometries and materials, this capacitance is on the order of a few to hundreds of femtofarads. It was possible to demonstrate that the ground plane, sometimes used, reduces the capacitance, but does not eliminate it completely. Possible noise sources are also discussed. The electrode tightness minimizes a possible source of mechanical noise due to variation of the coupling capacitances. Thermal control should also be ensured; the calculations showed that a temperature fluctuation as low as 7103 8C induces artifacts as high as the limit of quantification of K&thorn; in a typical electrophoretic condition, for which the technique has one of its highest sensitivities.