A multicommuted flow procedure for the determination of total and free cholesterol in eggs and human blood serum by chemiluminescence

Abstract

Um sistema de análises em fluxo com multicomutação com detecção por quimiluminescência foi desenvolvido para a determinação de colesterol livre e total sem alterações no módulo de análises. Colesterol estearase e colesterol oxidase foram imobilizadas em esferas de vidro via glutaraldeído/3-aminopropiletoxisilano e mini-colunas contendo as enzimas foram usadas para tratamento das amostras em linha. Ésteres de colesterol foram clivados a colesterol e ácidos graxos na coluna empacotada com colesterol estearase. No reator contendo colesterol oxidase, o analito foi convertido a 4-colesten-3-ona e peróxido de hidrogênio. A detecção foi baseada na quimiluminescência produzida por H 2 O 2 no sistema hexacianoferrato(III)-luminol. Os efeitos de variáveis químicas e hidrodinâmicas sobre o sinal de quimiluminescência foram avaliados. As curvas analíticas foram lineares entre 250 e 2500 mg L -1 e 500 e 4000 mg L -1 , para colesterol livre e total, respectivamente. Limites de detecção para ambos os analitos foram estimados em 60 mg L -1 (99,7% de confiança). A frequência de amostragem foi de 55 h -1 e o consumo de reagentes foi de 350 mg de luminol e 2,6 mg de hexacianoferrato(III) de potássio por determinação. O procedimento desenvolvido foi aplicado à determinação de colesterol em ovos e soro sanguíneo humano gerando resultados concordantes com o procedimento espectrofotométrico de referência com nível de confiança de 95%.A multicommuted flow-based procedure with detection by chemiluminescence for the determination of total and free cholesterol without changes in the flow manifold is proposed. Cholesterol esterase and cholesterol oxidase were both immobilized on glass beads via glutaraldehyde/(3-aminopropyl)-triethoxysilane and mini-columns containing the enzymes were used for online sample treatment. Cholesterol esters were cleaved to cholesterol and fatty acids at the packed reactor containing cholesterol esterase. The reactor containing cholesterol oxidase converted cholesterol to cholest-4-en-3-one also yielding hydrogen peroxide. Detection was based on the chemiluminescence produced by H 2 O 2 in the hexacyanoferrate(III)-luminol system. Influence of both chemical and hydrodynamic variables on the chemiluminescence signals were investigated. The analytical curves were linear from 250 to 2500 mg L -1 and from 500 to 4000 mg L -1 , for free and total cholesterol, respectively. Detection limits for both analytes were estimated as 60 mg L -1 at 99.7% confidence level. The sampling rate was 55 h -1 and reagent consumption was 350 mg of luminol and 2.6 mg of potassium hexacyanoferrate(III) per determination. The procedure developed was successfully applied for determination of cholesterol in eggs and in human blood serum with results in agreement with the reference spectrophotometric method at the 95% confidence level.

Development of a high sensitive automatic setup for screening of microcystins in surface waters by employing a LED-based photometric detector

“…The solution handling setup comprised a homemade syringe pump that was driven by a step-motor of 48 step per revolution; plastic syringe, 5.0 mm inner diameter and 50 mm long; a brass screw 5 cm long, 10 mm diameter and thread of 1.0 mm per revolution; a 161P011 pinch solenoid valve normally closed and a 161P021 pinch solenoid valve normally open (NResearch, 267 Fairfield Ave, West Caldwell, NJ, USA); a regulated power supply (12 V, 3 A) to feed the step-motor and the solenoid valves; two homemade digital interfaces [33] coupled to the output ports A and B of the PCL711 interface card to drive the step-motor, solenoid valves and LEDs; a homemade glass flow cell, 40 mm optical path long and 1.2 mm inner diameter (inner volume 46 L); three transistors BC547.…”

“…Sequential injection analysis (SIA) [21][22][23], multisyringe flow injection analysis (MSFIA) [24,25], and multicommuted flow analysis (MCFA) [26][27][28][29] approaches afford facilities for developing automated analytical procedures using lower volumes of sample and reagent solution than those based on the usual flow injection analysis (FIA) [27,28]. In this work, the facilities afforded by the MCFA approach [32][33][34], is employed to develop a setup for microcystin screening in surface water. A setup, including a manifold for handling sample solution and a LED-based photometer, is designed to allow three sampling runs to be performed using a sample volume of 200 L, in such a way that low levels of microcystin could be detected.…”

DNA-stabilized silver nanoclusters with guanine-enhanced fluorescence as a novel indicator for enzymatic detection of cholesterol

“…To date, many different methods have been used in the determination of cholesterol, such as electrochemical, colorimetric, uorescent and chemiluminescent detection. [9][10][11][12][13] However, each method has its own pros and cons with respect to the limits of the strategy and the apparatus used. Therefore, it is of considerable interest to develop more useful methods for the assay of cholesterol.…”

A LED based photometer for solid phase photometry: zinc determination in pharmaceutical preparation employing a multicommuted flow analysis approach

“…[10][11][12] Multicommuted flow analysis process affords facilities to design active flow setup constituted by solenoid valves, which have been assembled to work as an independent commutator unit, thus allowing facilities to handle sample and reagent solutions controlled by microcomputer. [8][9][10][11]13,14 Zinc is present in all living cells as a constituent of several molecules involved in proteins, lipids and carbohydrates metabolism, as well as several enzymes. 15,16 In this sense, zinc is considered as an essential element for all animals, including humans.…”