An integrated microfluidic-based biosensor using a magnetically controlled MNPs-enzyme microreactor to determine cholesterol in serum with fluorometric detection.

This work provides a microfluidic-based biosensor to determine total cholesterol in serum based on integrating the reaction/detection zone of a microfluidic chip of a magnetically retained enzyme microreactor (MREµR) coupled with the remote fluorometric detection through a bifurcated fiber-optic bundle (BFOB) connected with a conventional spectrofluorometer. The method is based on developing the enzymatic hydrolysis and oxidation of cholesterol at microscale size using both enzymes (cholesterol esterase (ChE) and cholesterol oxidase (ChOx)) immobilized on magnetic nanoparticles (MNPs). The biocatalyst reactions were followed by monitoring the fluorescence decreasing by the naphtofluorescein (NF) oxidation in the presence of the previous H2O2 formed. This microfluidic biosensor supposes the physical integration of a minimal MREµR as a bioactive enzyme area and the focused BFOB connected with the spectrofluorometer detector. The MREµR was formed by a 1 mm length of magnetic retained 2:1 ChE-MNP/ChOx-MNP mixture. The dynamic range of the calibration graph was 0.005-10 mmol L-1, expressed as total cholesterol concentration with a detection limit of 1.1 µmol L-1 (r2 = 0.9999, sy/x = 0.03, n = 10, r = 3). The precision expressed as the relative standard deviation (RSD%) was between 1.3 and 2.1%. The microfluidic-based biosensors showed a sampling frequency estimated at 30 h-1. The method was applied to determine cholesterol in serum samples with recovery values between 94.8 and 102%. The results of the cholesterol determination in serum were also tested by correlation with those obtained using the other two previous methods.

Study of the inhibition effects on glutathione peroxidase immobilized on MNPs using a stopped-flow microfluidic system.

A stopped-flow microfluidic system to monitor glutathione peroxidase (GPx) activity and evaluate potential inhibitors of the enzyme has been developed based on the integration of the microfluidic chip in the reaction/detection zone. This integration supposes the physical alignment at the optimal location of the microfluidic channel, both the magnetically retained enzyme microreactor (MREµR) and the remote luminescence detection using a focused bifurcated fiber optic bundle (BFOB) connected to a conventional spectrofluorometer detector. The method is based on the coupling of two competitive oxidative chemical reactions, in which glutathione (GSH) and homovanillic acid (HVA) competed for their interaction with hydrogen peroxide in the presence of the magnetically retained GPx-MNPs. The biocatalytic reaction was followed by monitoring the fluorescence of the biphenyl-HVA dimer formed. The dynamic range of the calibration graph was 0.45-10 µmol L-1, expressed as GSH concentration with a detection limit of 0.1 µmol L-1 (r2 = 0.9954, n = 10, r = 3). The precision expressed as the relative standard deviation (RSD%) was between 0.5 and 3.9%. The stopped-flow microfluidic system showed a sampling frequency of 25 h-1. The method was applied to the study of GPx inhibition provided by three inhibitory compounds, two metallic ions Hg(II) and Cu(II) and t-butyl hydroperoxide, and their presence in liquid samples, as water, milk, and edible oil. Recovery values between 88.7 and 99.4% were achieved in all instances.

Usefulness of Hybrid Magnetoliposomes for Aminoglycoside Antibiotic Residues Determination in Food Using an Integrated Microfluidic System with Fluorometric Detection.

A new microfluidic approach using hybrid magnetoliposomes (h-MLs) containing hydrophobic magnetic nanoparticles (Fe3O4@AuNPs-C12SH) and encapsulated N-acetylcysteine has been developed in this research to determine aminoglycoside antibiotic (AAG) residues in food using o-phthalaldehyde. Four AAGs, kanamycin, streptomycin, gentamicin, and neomycin, have been used as model analytes. The h-MLs have been used for reagent preconcentration and were retained using an external electromagnet device in the reaction/detection zone in a microfluidic system, inserted into the sample chamber of a conventional fluorimeter. The formation of a fluorescent isoindole derivate caused an increase in the luminescence signal, which was proportional to the analyte concentration. The dynamic range of the calibration graph was 0.1-1000 µmol L-1, expressed as AAG concentration, with an 8.7 nmol L-1 limit of detection for kanamycin and a sampling frequency of 8 h-1. The method was applied to determine AAG residues in milk and meat samples with recovery values between 87.2 and 107.4%.

Luminescence continuous flow system for monitoring the efficiency of hybrid liposomes separation using multiphase density gradient centrifugation.

A method for monitoring the efficiency of the hybrid magnetoliposomes (h-MLs) separation using multiphase density gradient centrifugation (MDGC) coupled with a continuous flow system (CFS) is described. Several h-MLs suspensions containing hydrophobic magnetic gold nanoparticles (Fe3O4@AuNPs-C12SH) and different fluorophores encapsulated have been synthesized using the rapid solvent evaporation (RSE) method. The MDGC system was prepared using a non-linear multiphase density gradient formed with a bottom layer with 100% (v/v) sucrose solution and six layers containing a mixture of sucrose solution (with concentrations ranged between 10 and 55% v/v), and fixed concentrations of ficoll (30% v/v) and percoll (15% v/v) solutions. The density gradient profile was previously stabilized using a relative centrifugal force (RCF) of 4480×g for 30 min. The synthesized h-MLs were added to the density gradient profile and separated by centrifugation at 2520×g for 20 min. The efficiency of the separation procedure was tested, aspirating the separated extract into the CFS and lysing liposomes before their translation to the detector introducing surfactant solutions. The luminescence signals provided by the release of the encapsulated fluorophores and other materials provided the distribution status of the liposomes in each density gradient stage. The monitoring of the different samples revealed four different fractions (MLs, h-Ls, h-MLs, and non-encapsulated fluorophores) for each separated h-MLs. Additional information on the h-MLs has also been acquired by confocal microscopy.

Separation and characterization of liposomes using asymmetric flow field-flow fractionation with online multi-angle light scattering detection.

Liposomes, mainly formed by phospholipids and cholesterol that entrapped different compounds, were separated and characterized using asymmetric flow field-flow fractionation (AF4) coupled with a multi-angle light scattering detector (MALS). AF4 allows the separation of liposomes according to their hydrodynamic size, and the particle size can be estimated directly by their elution time. Besides, different synthesized liposome suspensions of liposomes with different species encapsulated in different places in liposomes were prepared with analytical purposes to be studied. These liposomes were: empty liposomes (e-Ls), magnetoliposomes (MLs) with Fe3O4@AuNPs-C12SH inside the lipid bilayer, and long-wavelength fluorophores encapsulated into the aqueous cavity of liposomes (Ls-LWF). The optimization process of the variables that affect the fractionation has been established. The separation effectiveness has been compared with the results achieved with a photon-correlation spectroscopy analyzer based on dynamic light scattering (DLS) and transmission electron microscopy (TEM), used in self-assembly structures characterization. In all cases, three different classes of liposomes have been obtained; two are commonly appaired in all studied samples, while only a third class is characteristic for each of the liposomes. This mean that the proposed methodology could be used for identifying liposomes according to the encapsulated material.

Integration of a microfluidic system into a conventional luminescence detector using a 3D printed alignment device.

A useful 3D printed device for the inside microfluidic integration into a conventional optical detector has been developed. The coupling system supposes the complete integration of a microfluidic device inside the sample compartment of a conventional spectrofluorimeter. For this purpose, a commercial chip-holder, including a microfluidic chip, was anchored inside the detector using a "lab-built" 3D printing alignment prototype. The variables affecting the position of the 3D printed device, such as horizontal and vertical and rotary angles, were optimized. The usefulness of the microfluidic integration system has been tested using an organized suspension of separated hybrid magnetoliposomes containing nanomaterials that were previously separated using a multiphase density gradient centrifugation (MDGC) method. The whole integration system consists of three well-established parts: the impulsion unit, the displacement unit, and the microfluidic chip. The impulsion unit is formed by two syringe pumps, which propel under microflow-rate regime the solutions through to the microfluidic system. The first fluid incorporates an immiscible solution that provides the solution which fills positive oil/water (O/W) displacement unit. In this unit, the previously organized MDGC suspension, which includes different liposome populations, was layer-by-layer displaced to a y-mixer microfluidic chip. The separation content merges with the second solution propelled by the other syringe pump. This solution incorporates a surfactant that promotes the liposome lysis. The novelty supposes the easy incorporation of a 3D printer alignment device, which facilitates the incorporation of the microfluidic channel focused into the optical pathway of the luminescence detector. Graphical abstract.

Usefulness of magnetically-controlled MNPs-enzymes microreactors for the fluorimetric determination of total cholesterol in serum.

A new dynamic method containing a magnetically retained enzyme reactor (MRER) located in the reaction/detection zone of a flow injection (FI) system, has been used for the determination of total cholesterol in serum samples. The MRER was formed by a mixture ratio of 2/1 of immobilized enzymes cholesterol esterase (ChE) and cholesterol oxidase (COx) on magnetic nanoparticles (MNPs). The analytical signal is based on the fluorescence decreasing of the fluorophore naphtofluorescein (NF) due to its oxidation by the H2O2 formed in the enzymatic reactions. The dynamic range of the calibration graph was 1.55-100 mmol L-1 expressed as total cholesterol concentration (r2 = 0.9995, n = 5, r = 3), and the detection limit was 0.65 mmol L-1. The precision expressed as relative standard deviation (RSD %) was in the range of 4.7 and 0.6%. The method showed a sampling frequency of 10 h-1 and this method was applied to the determination of cholesterol in serum samples. The results were compared with those obtained using a previous automated clinical analyzer (ILab 600 analyzer). Also, recovery values ranging between 88.5 and 101.5% were achieved.