Molecularly imprinted magnetic nanoparticles as tunable stationary phase located in microfluidic channel for enantioseparation

A microfluidic device integrated with molecularly imprinted magnetic nanoparticles as stationary phase was designed for rapid enantioseparation by capillary electrochromatography. The nanoparticles were synthesized by the co-polymerization of methacrylic acid and ethylene glycol dimethacrylate on 3-(methacryloyloxy)propyltrimethoxysilane-functionalized magnetic nanoparticles (25-nm diameter) in the presence of template molecule, and characterized with infrared spectroscopy, thermal gravimetric analysis, and transmission electron microscope. The imprinted nanoparticles (200-nm diameter) could be localized as stationary phase in the microchannel of microfluidic device with the tunable packing length by the help of an external magnetic field. Using S-ofloxacin as the template molecule, the preparation of imprinted nanoparticles, the composition and pH of mobile phase, and the separation voltage were optimized to obtain baseline separation of ofloxacin enantiomers within 195 s. The analytical performance could be conveniently improved by varying the packing length of nanoparticles zone, showing an advantage over the conventional packed capillary electrochromatography. The linear ranges for amperometric detection of the enantiomers using carbon fiber microdisk electrode at +1.0 V (vs. Ag/AgCl) were from 1.0 to 500 μM and 5.0 to 500 μM with the detection limits of 0.4 and 2.0 μM, respectively. The magnetically tunable microfluidic device could be expanded to localize more than one kind of template-imprinted magnetic nanoparticles for realizing simultaneous analysis of different kinds of chiral compounds.     

Composite poly(dimethylsiloxane)/glass microfluidic system with an immobilized enzymatic particle-bed reactor and sequential sample injection for chemiluminescence determinations

A three-layer poly(dimethylsiloxane) (PDMS)/glass microfluidic system for performing on-chip solid-phase enzymatic reaction and chemiluminescence (CL) reaction was used for the determination of glucose as a model analyte. A novel method for the immobilization of controlled-pore-glass based reactive particles on PDMS microreactor beds was developed, producing an on-chip solid-phase reactor that featured large reactive surface and low flow impedance. Efficient mixing of reagent/sample/carrier streams was achieved by incorporating chaotic mixer structures in the microfluidic channels. A conventional sequential injection (SI) system was adapted for direct coupling with the microfluidic system, and combined with hydrostatic delivery of reagents to achieve efficient and reproducible sample introduction at 10 μl levels. A detection limit of 10 μM glucose (3σ), and a precision of 3.1% RSD (n=7, 0.2 mM glucose) were obtained using the SI-microfluidic-CL system integrated with a glucose oxidase (GOD) reactor. Carryover was <5% at a throughput of 20 samples/h.     

Microflow photo-radical reaction using a compact light source: application to the Barton reaction leading to a key intermediate for myriceric acid A

The Barton reaction (nitrite photolysis) of a steroidal substrate 1, to give 2, a key intermediate for the synthesis of myriceric acid A, an endothelin receptor antagonist, was successfully carried out in a continuous microflow system using a Pyrex glass-covered stainless-steel microreactor having a one lane microchannel (Type A: 1000 μm width, 107 μm depth, 2.2 m length). We found that using a low-power black light (peak wavelength: 352 nm) and UV-LED light (peak wavelength: 365 nm) as the light source will suffice for the Barton reaction, creating a compact energy-saving photo-microreaction system. A multi-gram-scale production was attained using a multi-lane microreactor (Type B: 1000 μm width, 500 μm depth, 0.5 m length, 16 lanes) in conjunction with a black light.     

Glutathione-capped CdTe quantum dots for the sensitive detection of glucose

A simple and sensitive assay system for glucose based on the glutathione (GSH)-capped CdTe quantum dots (QDs) was developed. GSH-capped CdTe QDs exhibit higher sensitivity to H₂O₂ produced from the glucose oxidase catalyzed oxidation of glucose, and are also more biocompatible than other thiols-capped QDs. Based on the quenching of H₂O₂ on GSH-capped QDs, glucose can be detected. The detection conditions containing reaction time, the concentration of glucose oxidase and the sizes of QDs were optimized and the detection limits for glucose was determined to be 0.1 μM; two detection ranges of glucose from 1.0 μM to 0.5 mM and from 1.0 mM to 20 mM, respectively were obtained. The detection limit was almost a 1000 times lower than other QDs-based optical glucose sensing systems. The developed glucose detection system was simple and facile with no need of complicated enzyme immobilization and modification of QDs.     

Electrochemical detection of glucose from whole blood using paper-based microfluidic devices

Electrochemical paper-based analytical devices (ePADs) with integrated plasma isolation for determination of glucose from whole blood samples have been developed. A dumbbell shaped ePAD containing two blood separation zones (VF2 membranes) with a middle detection zone was fabricated using the wax dipping method. The dumbbell shaped device was designed to separate plasma while generating homogeneous flow to the middle detection zone of the ePAD. The proposed ePADs work with whole blood samples with 24–60% hematocrit without dilution, and the plasma was completely separated within 4 min. Glucose in isolated plasma separated was detected using glucose oxidase immobilized on the middle of the paper device. The hydrogen peroxide generated from the reaction between glucose and the enzyme pass through to a Prussian blue modified screen printed electrode (PB-SPEs). The currents measured using chronoamperometry at the optimal detection potential for H₂O₂ (−0.1 V versus Ag/AgCl reference electrode) were proportional to glucose concentrations in the whole blood. The linear range for glucose assay was in the range 0–33.1 mM (r² = 0.987). The coefficients of variation (CVs) of currents were 6.5%, 9.0% and 8.0% when assay whole blood sample containing glucose concentration at 3.4, 6.3, and 15.6 mM, respectively. Because each sample displayed intra-individual variation of electrochemical signal, glucose assay in whole blood samples were measured using the standard addition method. Results demonstrate that the ePAD glucose assay was not significantly different from the spectrophotometric method (p = 0.376, paired sample t-test, n = 10).     

Development and evaluation of electrochemical glucose enzyme biosensors based on carbon film electrodes

Electrochemical glucose enzyme biosensors have been prepared on carbon film electrodes made from carbon film electrical resistors. Evaluation and characterisation of these electrodes in phosphate buffer saline solution has been carried out with and without pretreatment by cycling in perchloric acid or at fixed applied potential. Both pretreatments led to a reduction in the carbon surface oxidation peak and enabled better detection of hydrogen peroxide in the pH range of 5-7. Glucose oxidase enzyme was immobilised on the carbon surface by mixing with glutaraldehyde, bovine serum albumin and with and without Nafion. The performance of these two types of electrode was similar, that containing Nafion being more physically robust. Linear ranges were up to around 1.5 mM, with detection limits 60 μM, and pretreatment of the carbon film electrode at a fixed potential of +0.9 V versus SCE for 5 min was found to be the most beneficial. Michaelis-Menten constants between 5 mM and 10 mM were found under the different experimental conditions. Coating the immobilised enzyme layer with a thin layer of Nafion was found to give similar results in the determination of glucose to mixing it but with benefits against interferences for the analysis of complex matrices, such as wine. Potentialities, for a short-term-use or disposable sensors, are indicated.     

A novel amperometric biosensor based on NiO hollow nanospheres for biosensing glucose

NiO hollow nanospheres were synthesized by controlled precipitation of metal ions with urea using carbon microspheres as templates, which were for the first time adopted to construct a novel amperometric glucose biosensor. Glucose oxidase was immobilized on the surface of hollow nanospheres through chitosan-assisted cross-linking technique. Due to the high specific active sites and high electrocatalytic activity of NiO hollow nanospheres, the constructed glucose biosensors exhibited a high sensitivity of 3.43 μA/mM. The low detection limit was estimated to be 47 μM (S/N = 3), and the Michaelis-Menten constant was found to be 7.76 mM, indicating the high affinity of enzyme on NiO hollow nanospheres to glucose. These results show that the NiO hollow nanospheres are a promising material to construct enzyme biosensors.     

Size dependence of SiO2 particles enhanced glucose biosensor

A wide size range of SiO₂ particles were synthesized and were used as enzyme immobilization carriers to fabricate glucose biosensors. The size of the particles was in the range of 17-520 nm. These biosensors could be operated under physiological conditions (0.1 M phosphate buffer, pH 7.2). Particle size could affect the performance of SiO₂ modified glucose biosensors drastically. The smaller particles had higher performance. The smallest SiO₂ modified biosensor could work well in the glucose concentration range of 0.02-10 mM with a correlation coefficient of 0.9993. Its sensitivity was 2.08 μA/mM and the detection limit was 1.5 μM glucose.     

The Barton reaction using a microreactor and black light. Continuous-flow synthesis of a key steroid intermediate for an endothelin receptor antagonist

The Barton reaction (nitrite photolysis) of a steroidal substrate 1, to give 2, a key intermediate for the synthesis of an endothelin receptor antagonist, was successfully carried out in a continuous microflow system using a pyrex glass-covered stainless-steel microreactor having a microchannel (Type A: 1000 μm width, 107 μm depth, 2.2 m length). We found that a 15 W black light (peak wavelength: 352 nm) as the light source, suffices for the Barton reaction, creating a compact photo-micro reaction system. Multi-gram scale production was attained using two serially connected, multi-lane microreactors (Type B).     

Selective and sensitive electrochemical detection of glucose in neutral solution using platinum-lead alloy nanoparticle/carbon nanotube nanocomposites

Electrodeposition of Pt-Pb nanoparticles (PtPbNPs) to multi-walled carbon nanotubes (MWCNTs) resulted in a stable PtPbNP/MWCNT nanocomposite with high electrocatalytic activity to glucose oxidation in either neutral or alkaline medium. More importantly, the nanocomposite electrode with a slight modification exhibited high sensitivity, high selectivity, and low detection limit in amperometric glucose sensing at physiological neutral pH (poised at a negative potential). At +0.30 V in neutral solution, the nanocomposite electrode exhibited linearity up to 11 mM of glucose with a sensitivity of 17.8 μA cm⁻² mM⁻¹ and a detection limit of 1.8 μM (S/N = 3). Electroactive ascorbic acid (0.1 mM), uric acid (0.1 mM) and fructose (0.3 mM) invoked only 23%, 14% and 9%, respectively, of the current response obtained for 3 mM glucose. At −0.15 V in neutral solution, the electrode responded linearly to glucose up to 5 mM with a detection limit of 0.16 mM (S/N = 3) and detection sensitivity of ∼18 μA cm⁻² mM⁻¹. At this negative potential, ascorbic acid, uric acid, and fructose were not electroactive, therefore, not interfering with glucose sensing. Modification of the nanocomposite electrode with Nafion coating followed by electrodeposition of a second layer of PtPbNPs on the Nafion coated PtPbNP/MWCNT nanocomposite produced a glucose sensor (poised at −0.15 V) with a lower detection limit (7.0 μM at S/N = 3) and comparable sensitivity, selectivity and linearity compared to the PtPbNP/MWCNT nanocomposite. The Nafion coating lowered the detection limit by reducing the background noise, while the second layer of PtPbNPs restored the sensitivity to the level before Nafion coating.     

Microfabricated bioreactor chips for immobilised enzyme assays

A microfabricated device is reported that has been designed to permit the in situ packing of a section of channel with enzyme immobilised onto controlled pore glass (CPG). It is fabricated from glass and polydimethylsiloxane and to prevent dead volumes, has dedicated channels for packing the reactor. The device has the advantage of being simple in design, the flow through enzyme reactor channel being simply a widened section of the analyte channel. The system is suitable for both hydrodynamic and electro-osmotic pumping, and is designed such that when the packing is exhausted it can be repacked. Controlled pore glass provides a reproducible none swelling, high porosity medium onto which the enzyme could be immobilised. The large particle size meant that it was vital to optimise the immobilisation procedure in order to achieve acceptable enzyme activity. The microfabricated device was developed with two enzymes of different molecular masses; alkaline phosphatase and xanthine oxidase. The pore size of the CPG was found to be very important for xanthine oxidase, where the 697 Å pore size (120-200 mesh) CPG was found to give the highest activity (18-20% activity retained after immobilisation). The microfabricated device was used for the assay of p-nitrophenyl phosphate and hypoxanthine with spectrophotometric detection at 405 and 470 nm, respectively. The limits of detection were 5 and 8 μM, respectively.     

Determination of beta-lactam antibiotics in milk using micro-flow chemiluminescence system with on-line solid phase extraction

In this paper, a chemiluminescence (CL) micro-flow system combined with on-line solid phase extraction (SPE) is presented for determination of β-lactam antibiotics (penicillin, cefradine, cefadroxil, cefalexin) in milk. It is based on the enhancement effect of β-lactam antibiotics on the luminol-K₃Fe(CN)₆ CL system. The micro-flow system was fabricated from two polymethyl methacrylate (PMMA) plates (50 mm × 40 mm × 5 mm) with the microchannels of 200 μm wide and 150 μm deep. C₁₈-modified silica gel was packed into the microchannel (length: 10 mm; width: 1 mm; depth: 500 μm) to serve as SPE device. Extraction and preconcentration of the analytes were carried out using on-line SPE micro-flow system and the selectivity of CL detection was improved. The detection limits were 0.5 μg mL⁻¹ of penicillin, 0.04 μg mL⁻¹ of cefradine, 0.08 μg mL⁻¹ of cefadroxil and 0.1 μg mL⁻¹ of cefalexin. The proposed method was also applied to analyze the β-lactam antibiotics in milk. Experimental results were in good agreement with those obtained by high performance liquid chromatography (HPLC) method with UV detection.     

PdNi- and Pd-coated electrodes prepared by electrodeposition from ionic liquid for nonenzymatic electrochemical determination of ethanol and glucose in alkaline media

Nonenzymatic electrochemical determination of ethanol and glucose was respectively achieved using PdNi- and Pd-coated electrodes prepared by electrodeposition from the novel metal-free ionic liquid (IL); N-butyl-N-methylpyrrolidinium dicyanamide (BMP-DCA). BMP-DCA provided an excellent environment and wide cathodic limit for electrodeposition of metals and alloys because many metal chlorides could dissolve in this IL where the reduction potentials of Pd(II) and Ni(II) indeed overlapped, leading to the convenience of potentiostatic codeposition. In aqueous solutions, the reduction potentials of Pd(II) and Ni(II) are considerably separated. The bimetallic PdNi coatings with atomic ratios of ∼80/20 showed the highest current for ethanol oxidation reaction (EOR). Ethanol was detected by either cyclic voltammetry (CV) or hydrodynamic amperometry (HA). Using CV, the dependence of EOR peak current on concentration was linear from 4.92 to 962 μM with a detection limit of 2.26 μM (σ = 3), and a linearity was observed from 4.92 to 988 μM using HA (detection limit 0.83 μM (σ = 3)). The Pd-coated electrodes prepared by electrodeposition from BMP-DCA showed electrocatalytic activity to glucose oxidation and CV, HA, and square-wave voltammetry (SWV) were employed to determine glucose. SWV showed the best sensitivity and linearity was observed from 2.86 μM to 107 μM, and from 2.99 mM to 10.88 mM with detection limits of 0.78 μM and 25.9 μM (σ = 3), respectively. For glucose detection, the interference produced from ascorbic acid, uric acid, and acetaminophen was significantly suppressed, compared with a regular Pt disk electrode.     

Electro-oxidation of glucose at self-assembled monolayers incorporated by copper particles

A novel copper incorporated self-assembled monolayers (SAMs) modified gold electrode (Cu/SAMs) for determination of glucose was developed by electrodepositing Cu particles on the SAMs of hexanethiol. The scanning electron microscopic (SEM) images showed that copper formed orbicular particles of nanosizes on the SAMs, which was much different from the fractal-like particles of copper formed at gold electrode. The Cu/SAMs film electrode exhibited high sensitivity to glucose oxidation and depressed responses towards some interferents of glucose in blood like uric acid and ascorbic acid. Under optimal working conditions, the oxidation current of glucose was proportional to the concentration of glucose in the range from 3.0 μM to 10 mM by amperometry with a low detection limit of 0.7 μM glucose (S/N = 3). This electrode was successfully applied to the determination of glucose in rat blood and the results were satisfactory.     

Fluorometric method for the determination of hydrogen peroxide and glucose with Fe3O4 as catalyst

In this paper, we utilized the instinct peroxidase-like property of Fe₃O₄ magnetic nanoparticles (MNPs) to establish a new fluorometric method for determination of hydrogen peroxide and glucose. In the presence of Fe₃O₄ MNPs as peroxidase mimetic catalyst, H₂O₂ was decomposed into radical that could quench the fluorescence of CdTe QDs more efficiently and rapidly. Then the oxidization of glucose by glucose oxidase was coupled with the fluorescence quenching of CdTe QDs by H₂O₂ producer with Fe₃O₄ MNPs catalyst, which can be used to detect glucose. Under the optimal reaction conditions, a linear correlation was established between fluorescence intensity ratio I₀/I and concentration of H₂O₂ from 1.8 × 10⁻⁷ to 9 × 10⁻⁴ mol/L with a detection limit of 1.8 × 10⁻⁸ mol/L. And a linear correlation was established between fluorescence intensity ratio I₀/I and concentration of glucose from 1.6 × 10⁻⁶ to 1.6 × 10⁻⁴ mol/L with a detection limit of 1.0 × 10⁻⁶ mol/L. The proposed method was applied to the determination of glucose in human serum samples with satisfactory results.     

Long pathlength, three-dimensional absorbance microchip

A long pathlength, three-dimensional U-type flow cell was microfabricated and evaluated for improved absorbance detection on a glass microdevice. A small diameter hole (75 μm) was laser etched in a thin glass substrate whose thickness (100 μm) defined much of the pathlength of the cell. This substrate was thermally bonded and sandwiched between two different glass substrates. The top substrate contained a typical injection cross and separation microchannel. Projecting out of the plane of the separation device was a 126 μm pathlength flow cell as defined by the laser etched hole and the attached microchannels. The flow cell was connected to a microchannel on the bottom substrate that led to a waste reservoir. The planar, flat windows on the top and bottom of this device made light introduction and collection a simple matter using a light emitting diode (LED) and microscope objective. The experimentally obtained detection limit for rhodamine B was determined to be 0.95 μM, which is nearly identical to the theoretical limit calculated by Beer's Law. A separation of three fluorescent dyes was performed, and direct comparisons were made between the transmittance changes through the narrow pathlength separation microchannel and the adjacent long pathlength, three-dimensional U-type flow cell.     

A glucose biosensor using methyl viologen redox mediator on carbon film electrodes

A new methyl viologen-mediated amperometric enzyme electrode sensitive to glucose has been developed using carbon film electrode substrates. Carbon film electrodes from resistors fabricated by pyrolytic deposition of carbon were modified by immobilization of glucose oxidase through cross-linking with glutaraldehyde in the presence of bovine serum albumin. The mediator, methyl viologen, was directly immobilised with the enzyme together with Nafion cation-exchange polymer. The electrochemistry of the glucose oxidase/methyl viologen modified electrode was investigated by cyclic voltammetry and by electrochemical impedance spectroscopy. The biosensor response to glucose was evaluated amperometrically; the detection limit was 20 μM, the linear range extended to 1.2 mM and the reproducibility of around 3%. When stored in phosphate buffer at 4 °C and used every day, the sensor showed good stability over more several weeks.     

Pretreatment-free fast ultraviolet detection of melamine in milk products with a disposable microfluidic device

A new method for sensitive and fast screening of melamine (MEL) in milk products was developed with a low-cost disposable microfluidic device coupled with ultraviolet (UV) detection. This method avoided the need of sample pretreatment prior to the separation process, thus was simple and green. Due to the advantages of the device and fracture sampling technique, milk sample could be directly sampled through the fracture to achieve baseline separation from amino acids, and proteins in the sample did not interfere with the detection. Using 20 mM phosphate running buffer (pH 9.0), a sampling time of 3 s at +180 V and a separation voltage of +1800 V (240 V/cm), this method could detect MEL in milk within 75 s. At the detection wavelength of 202 nm, the linear range for MEL was from 1.0 to 100 μg mL⁻¹ with a detection limit of 0.23 μg mL⁻¹ (S/N = 3). The novel protocol had been successfully used to screen MEL in milk samples with recovery more than 82%. The environmentally friendly methodology for pretreatment-free sensitive screening of MEL provided promising applications in monitoring the quality of different foods.     

Determination of mannitol and three sugars in Ligustrum lucidum Ait. by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection has been developed for the separation and determination of mannitol, sucrose, glucose, and fructose in Ligustrum lucidum Ait. for the first time. Effects of several important factors such as the concentration of NaOH, separation voltage, injection time, and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 300 μm diameter copper disc electrode at a working potential of +0.65 V (versus saturated calomel electrode (SCE)). The four analytes can be well separated within 13 min in a 40 cm length fused-silica capillary at a separation voltage of 12 kV in a 75 mM NaOH aqueous solution. The relation between peak current and analyte concentration was linear over about three orders of magnitude with detection limits (S/N = 3) ranging from 1 to 2 μM for all analytes. The proposed method has been successfully applied to monitor the mannitol and sugar contents in the plant samples at different growth stages with satisfactory assay results.     

Photochemical immobilization of protein on the inner wall of a microchannel and Its application in a glucose sensor

A new protein immobilization technique has been developed for patterning enzymes in a specific position inside a microchannel. First, bovine serum albumin (BSA) was adsorbed onto the internal surface of a polydimethylsiloxane microchannel. The microchannel was then filled with the conjugate solution of a photoreactive cross-linker, 4-azido-2,3,5,6-tetrafluorobenzoic acid succinimidyl ester (ATFB-SE), and an enzyme, horseradish peroxidase (HRP). An irradiation by a He-Cd laser activated the azido group of the conjugates and these conjugates became covalently attached to the adsorbed BSA on the microchannel. The enzyme turnover was observed from only the HRP zone. This technique was successfully applied to the enzymatic glucose sensor. Glucose oxidase (GOD) and HRP were sequentially patterned in a single microchannel, i.e., the HRP zone was located downstream from the GOD zone. The calibration curve of a glucose standard solution was linear over the range of 0-128 μM with a correlation coefficient of 0.993. Compared to the traditional method using a 96-well microtiter plate, the present technique on the microchip shortened the reaction time from 30 min to 4.8 s, i.e., to 1/375.