Integrated microfluidic systems for fluorescence monitoring rapid kinetic reactions in bioanalysis.

A stopped-flow microfluidic fluorimetric biosensor to monitor alkaline phosphatase (ALP) activity and evaluate the potential inhibitors has been developed, integrating a magnetically retained enzyme microreactor (MREµR) in the reaction/detection zone of the microfluidic chip. The integration supposed the alignment of the MREµR at the sample compartment of a conventional spectrofluorometer using a 3D-printed device. The analytical signal is based on the fluorescence decrease in the signal obtained in the dephosphorylation reaction of the substrate 4-methylumbelliferone phosphate (4-MUP) by the retained ALP-MNPs in an alkaline medium caused by sulfonamides. The excitation and emission wavelengths to monitor the reaction were 363 and 444 nm, respectively. Three sulfonamides, acetazolamide, furosemide, and sulfasalazine, have been used as model analytes. The front-face operating mode of the spectrofluorometer was used to acquire the instrumental signals. The influence of the rotation angle of the microfluidic device on the efficiency of the signal collection has also been studied, obtaining the signals with greater intensity at 75° from the excitation beam. The dynamic range of the calibration graph was 16.81-1111.22 µg mL-1, expressed as sulfonamide concentration, with a limit of detection of 5.04 µg mL-1 (R2 = 0.9989, n = 10, r = 3) for acetazolamide. The method was applied to determine sulfonamide residues in tap water and milk samples, with 88.9-98.7% recovery values. The results have been compared with those obtained using a commercial device connected to the spectrofluorometer, getting faster reaction kinetics.

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%.

Development of an aptamer-based SPR-biosensor for the determination of kanamycin residues in foods.

A biosensor in which an affinity reaction occurs in the sensitive microzone through the use of specific aptamers to determine kanamycin residues in agri-food samples has been developed. It is an irreversible and continuous flow aptameric biosensor (aptasensor) in which the signal variations are monitored by surface plasmon resonance (SPR) measurements based on the specific interaction of the aptamer with the antibiotic. The signal variation is proportional to the analyte concentration. Graphene is known for efficient binding of molecules with its π-electron system, so a monolayer of graphene prepared from chemical vapor deposition (CVD) has been compared to a multilayer of graphene made from reduced graphene oxide (rGO) for immobilization of the aptamer on the gold surface of the physicochemical transducer. The best results have been obtained with CVD graphene. The dynamic range was between 1 and 100 µmol L-1 of kanamycin concentration (r2 = 0.9981, n = 7, r = 4), with a limit of detection of 285 nmol L-1 and a sampling frequency of 6 h-1. The precision, expressed as relative standard deviation (RSD%), was established in the range of 1.49 and 3.89%, calculated for 1, 10, and 50 µmol L-1. The selectivity was studied applying the described method to determine other antibiotics, obtaining no significant difference in the analytical signal. The method was applied to determine kanamycin residues in milk samples with recovery values ranging between 90 and 96%.

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.

Automatic determination of coenzyme Q10 in food using cresyl violet encapsulated into magnetoliposomes.

A new type of magnetoliposomes (MLs), containing hydrophobic magnetic-gold nanoparticles and the long wavelength fluorophore cresyl violet, has been used for the determination of coenzyme Q10 (CoQ10). MLs were concentrated just before the detector, using a flow system and an external electromagnet device. The subsequent introduction of Triton X-100 and CoQ10 causes the MLs lysis and the cresyl violet oxidation, obtaining a decrease in the fluorescence signal. The dynamic range of the calibration graph was 0.03-0.50µmolL-1 CoQ10, and the detection limit was 0.008µmolL-1. The precision (relative standard deviation) was in the range of 1.3-4.5%. The method showed a sampling frequency of 12h-1 and was applied to the determination of CoQ10 in several food samples. The results were compared with those obtained using a previously described chromatographic method. Also, recovery values were in the range of 83.5-101.3%.

Fluorometric determination of alkaline phosphatase activity in food using magnetoliposomes as on-flow microcontainer devices.

Liposomes containing magnetic gold nanoparticles (AuNPs) and an enzymatic substrate (4-methylumbelliferyl-phosphate) have been used as on-flow microcontainers for reagent preconcentration in a flow injection method for the determination of alkaline phosphatase (ALP) activity. The dynamic range of the calibration graph was 6.4 × 10(-3)-0.25 U L(-1) ALP, and the detection limit was 1.9 × 10(-3) U L(-1). The precision, expressed as relative standard deviation (RSD%), was in the range of 0.7-2.4%. The overall method showed a sampling frequency of 10 h(-1). The method was applied to the determination of ALP in milk samples with recovery values ranging between 87.5 and 104.6%. The residual ALP activity in milk samples subjected to temperature treatments was also determined. The results obtained in the analysis of all milk samples were compared with those obtained by applying a previously described flow injection method.

Gold nanoparticle-biotinylated liposome hybrids as analytical reagents for biotin determination using a competitive assay and resonance light scattering detection.

The preparation of hybrid nanostructures formed by gold nanoparticles (AuNPs) into biotinylated liposomes and their analytical application are presented. The surface of negatively charged AuNPs was modified with 1-dodecanethiol and the NPs were encapsulated into biotinylated liposomes using the rapid solvent evaporation method. Liposomes were resized by both mechanical shaking and ultrasound treatments and filled liposomes were separated from empty liposomes using sucrose density gradient centrifugation. The analytical usefulness of AuNP-liposome hybrids as amplification probes for biotin determination was checked using the competitive affinity reaction based on the avidin-biotin interaction and biotilynated phospholipids for the synthesis of the liposome hybrids. The method was automatized using a flow system and measuring the resonance light scattering signal. The dynamic range of the calibration graph was 0.001-20 µg mL(-1), (r(2)=0.9998, n=14), with a detection limit of 0.3 ng mL(-1). The precision, expressed as relative standard deviation (RSD%), was lower than 5% and the sampling frequency was 9 h(-1). The approach has been applied to the determination of biotin in food samples, with recovery values ranging between 88.2 and 105.2%.

Luminescent determination of flavonoids in orange juices by LC with post-column derivatization with aluminum and terbium.

A new post-column derivatization system is described and applied to the determination of flavonoids in citric beverages after their separation by LC using a monolithic column. The derivatization involves the formation of the chelates of the analytes with aluminum (III) and terbium (III) in the presence of the surfactant SDS and the measurement of the terbium sensitized luminescence at lambda(ex) 360 and lambda(em) 545 nm. Naringin, hesperidin, quercetin, naringenin, and kaempferol have been chosen as analyte models. The large Stokes shift and the relatively long wavelength emission of terbium(III) can minimize interferences from background sample matrix, which usually emit at shorter wavelengths. Calibration graphs were constructed in the intervals 6.0-1700 ng/mL naringin, 9.8-1700 ng/mL hesperidin, 2.1-2000 ng/mL quercetin, 5.2-1500 ng/mL naringenin and 2.5-2000 ng/mL kaempferol, with regression coefficients higher than 0.9935 in all instances. The precision of the method, expressed as RSD%, was established at two concentration levels, with values of 1.3 and 4.7%, which corresponded to the minimal and maximal error zones of the calibration graphs. The practical usefulness of the method is demonstrated by the analysis of orange juices, which were diluted and directly injected into the chromatographic system, obtaining recoveries between 86.9 and 108.2%.

Analytical innovations in the detection of phenolics in wines.

A liquid chromatographic method with online photometric and luminescent detection for the determination of 18 phenolic compounds in wines is reported. Photometric detection is performed at four wavelengths, namely, 256, 280, 320, and 365 nm, using a diode array detection system. The luminescent detection is achieved by means of a postcolumn derivatization reaction of 10 of these compounds with terbium(III) in the presence of synergistic agents, such as ethylenediaminetetraacetic acid (EDTA) and n-octyltriphosphine oxide (TOPO). A micellar medium provided by the surfactants sodium dodecyl sulfate and Triton X-100 was used for the determination of the luminescent chelates at lambdaex 317 and lambdaem 545 nm. The long wavelength emission of lanthanide chelates can minimize interferences from background sample matrix, which usually emit at shorter wavelengths. The analytical features of the photometric and fluorometric methods, such as dynamic ranges of the calibration graphs, detection limits, and precision data, have been obtained. The practical usefulness of the developed methods is demonstrated by the analysis of Spanish and Italian wine samples (red, rosé, oloroso, and white), which were diluted and directly injected into the chromatographic system. The accuracy of both methods was checked by assaying a recovery study, which was performed at three different analyte levels for each type of sample.

Determination of fluoroquinolones in milk samples by postcolumn derivatization liquid chromatography with luminescence detection.

A liquid chromatography (LC) method with luminescence detection for the determination of eight quinolone antibiotics is reported. The system encompasses three consecutive steps: (a) chromatographic separation using reverse-phase mode (RP-LC), (b) postcolumn derivatization reaction, and (c) luminescence detection by monitoring fluorescence (FL) and time-resolved (TR) signals. The derivatization step is based on the reaction between quinolones and terbium(III) to form luminescent chelates, which were determined at lambda(ex) 340 and lambda(em) 545 nm (FL mode) or at lambda(ex) 281 and lambda(em) 545 nm (TR mode). Dynamic ranges of the calibration graphs, obtained with standard solutions of analytes and FL and TR modes, respectively, were 190-3500 and 316-2000 ng mL-1 for marbofloxacin, 8-3500 and 8.1-1500 ng mL-1 for ciprofloxacin, 6.2-3500 and 13-1500 ng mL-1 for danofloxacin, 7.4-3500 and 8.4-1500 ng mL-1 for enrofloxacin, 14-3500 and 20-2000 ng mL-1 for sarafloxacin, 12.5-3500 and 13.9-1200 ng mL-1 for difloxacin, 7.6-3500 and 13-3000 ng mL-1 for oxolinic acid, and 9-2000 and 130-3000 ng mL-1 for flumequine. Limit of detection values obtained using FL and TR modes, respectively, were 60 and 95 ng mL-1 for marbofloxacin, 2 and 2.4 ng mL-1 for ciprofloxacin, 1.9 and 3.9 ng mL-1 for danofloxacin, 2.2 and 2.5 ng mL-1 for enrofloxacin, 3.8 and 7 ng mL-1 for sarafloxacin, 4 and 4.2 ng mL-1 for difloxacin, 2.3 and 4 ng mL-1 for oxolinic acid, and 2.7 and 40 ng mL-1 for flumequine. The precision was established at two concentration levels of each analyte and expressed as the percentage of relative standard deviation with values ranging between 1.9 and 7.8%. The validation procedure for the analysis of samples was carried out using European Community recommendations, and the decision limit and detection capability were calculated for bovine whole milk. The method was applied to whole, semiskimmed, and skimmed milk samples spiked with the target analytes, and the recoveries ranged between 93.3 and 106.0%.