无筛板型固相萃取柱的制备及其在食品中苯甲酸的测定研究

结合固相萃取(SPE)盘与含支撑物的SPE柱技术,制备了一种新型的无筛板型固相萃取柱.以C18填料为例,以话梅样品为介质对其中的苯甲酸进行分析,并用传统固相萃取小柱平行比较;将SPE与HPLC-UV结合,考察了填料对简单介质中苯甲酸的最大吸附量及洗脱曲线,研究了新型SPE柱在实际应用中的分离纯化效果.结果表明,新型SPE柱对样品的吸附效果更好,规格为200 mg/3 mL的SPE柱对苯甲酸的吸附量达到0.951 mg,超过了传统柱的吸附量0.908 mg;其洗脱曲线与传统柱几乎重合;苯甲酸在1～100 mg/L浓度范围内线性关系良好, r=0.9999,用此SPE柱纯化后的样品加标回收率和相对误差分别在88.4%～102.3%和1.4%～2.9%之间.     

Microchip extraction of catecholamines using a boronic acid functional affinity monolith

A novel solid phase extraction microchip with a boronic acid functional affinity monolithic disc was developed in this work. Vinyl phenylboronic acid–ethylene glycol dimethacrylate co-polymer monoliths, which have pore sizes up to 20 μm, were investigated for extraction of catecholamines using adsorption and desorption studies in a batch system. Desorption yields of greater than 90% were achieved for catecholamines at pH 3 and below. Monolithic discs were then formed in chambers in borofloat glass microfluidic chips using in situ UV polymerization. Adsorption on the monolithic discs was performed via electrokinetic flow, with catecholamines determined via laser-induced native fluorescence (LINF) detection following electrokinetic elution. Microchips containing the boronic acid functional polymer discs worked well for extraction of catecholamines, providing greater than 100 fold concentration enrichment. This study demonstrated that a solid phase extraction microchip, containing an easily prepared monolith disc, will be useful for boronate affinity extraction of cis-diol containing compounds.     

Solid supports for micro analytical systems

The development of micro analytical systems requires that fluids are able to interact with the surface of the microfluidic chip in order to perform analysis such as chromatography, solid phase extraction, and enzymatic digestion. These types of analyses are more efficient if there are solid supports within the microfluidic channels. In addition, solid supports within microfluidic chips are useful in producing devices with multiple functionalities. In recent years there have been many approaches introduced for incorporating solid supports within chips. This review will explore several state of the art methods and applications of introducing solid supports into chips. These include packing chips with beads, incorporating membranes into chips, creating supports using microfabrication, and fabricating gels and polymer monoliths within microfluidic channels.     

Measurements of scattered light on a microchip flow cytometer with integrated polymer based optical elements

Flow cytometry is widely used for analyzing microparticles, such as cells and bacteria. In this paper, we report an innovative microsystem, in which several different optical elements (waveguides, lens and fiber-to-waveguide couplers) are integrated with microfluidic channels to form a complete microchip flow cytometer. All the optical elements, the microfluidic system, and the fiber-to-waveguide couplers were defined in one layer of polymer (SU-8, negative photoresist) by standard photolithography. With only a single mask procedure required, all the fabrication and packaging processes can be finished in one day. Polystyrene beads were measured in the microchip flow cytometer, and three signals (forward scattering, large angle scattering and extinction) were measured simultaneously for each bead. To our knowledge this is the first time forward scattered light and incident light extinction were measured in a microsystem using integrated optics. The microsystem can be applied for analyzing different kinds of particles and cells, and can easily be integrated with other microfluidic components.     

Integrated multilayer microfluidic device with a nanoporous membrane interconnect for online coupling of solid-phase extraction to microchip electrophoresis

An integrated microfluidic device was developed for online coupling of solid-phase extraction to microchip electrophoresis (chip SPE-CE). With a nanoporous membrane sandwiched between two PDMS substrates, SPE preconcentration and electrophoretic separation can be carried out in upper and lower fluidic layers, separately and sequentially. During the SPE process, the thin membrane can act as a fluid isolator to prevent intermixing between two fluidic channels. However, when a pulse voltage is applied, the membrane becomes a gateable interconnect so that a small plug of concentrated analytes can be online injected into the lower channel for subsequent separations. This multilayer design provides a universal solution to online SPE-CE hyphenation. Both electroosmotic flow and hydrodynamic pumps have been adopted for SPE operation. SPE was performed on a 2.5 mm long microcolumn, with two weirs on both sides to retain the C(18)-coated silica beads. Rhodamine 123 and FITC-labelled ephedrine were used to test the operational performance of the hyphenation system. High separation efficiency and thousand-fold signal enhancement were achieved.     

Integrated electrokinetically driven microfluidic devices with pH‐mediated solid‐phase extraction coupled to microchip electrophoresis for preterm birth biomarkers

Integration in microfluidics is important for achieving automation. Sample preconcentration integrated with separation in a microfluidic setup can have a substantial impact on rapid analysis of low‐abundance disease biomarkers. Here, we have developed a microfluidic device that uses pH‐mediated solid‐phase extraction (SPE) for the enrichment and elution of preterm birth (PTB) biomarkers. Furthermore, this SPE module was integrated with microchip electrophoresis for combined enrichment and separation of multiple analytes, including a PTB peptide biomarker (P1). A reversed‐phase octyl methacrylate monolith was polymerized as the SPE medium in polyethylene glycol diacrylate modified cyclic olefin copolymer microfluidic channels. Eluent for pH‐mediated SPE of PTB biomarkers on the monolith was optimized using different pH values and ionic concentrations. Nearly 50‐fold enrichment was observed in single channel SPE devices for a low nanomolar solution of P1, with great elution time reproducibility (<7% RSD). The monolith binding capacity was determined to be 400 pg (0.2 pmol). A mixture of a model peptide (FA) and a PTB biomarker (P1) was extracted, eluted, injected, and then separated by microchip electrophoresis in our integrated device with ∼15‐fold enrichment. This device shows important progress towards an integrated electrokinetically operated platform for preconcentration and separation of biomarkers.     

Novel approach for fritless capillary electrochromatography

At present, the main limitation for the further adoption of capillary electrochromatography (CEC) in the (routine) laboratory is caused by the lack of reproducible and stable columns. The main source of column instability is concentrated in the frits needed to retain the packed bed inside the CEC capillary. The sintering process used to prepare the frits can be rather problematic and irreproducible, particularly for small stationary phase particles and wide column diameters. Since the (surface) composition of the frits is different from the bulk stationary phase packing, different electroosmotic flow (EOF) velocities are generated. This effect is assumed to be primarily responsible for rapid column destruction. In this contribution, a novel approach for the preparation of fritless CEC capillaries is presented and evaluated. Using 5 microm Hypersil ODS particles, separation efficiencies in the range of 130,000-200,000 plates/m were obtained. In a 100 microm inner diameter packed column, electrical currents up to 50 microA could be tolerated without negative effects such as bubble formation. The prepared CEC columns were found to be stable and could easily be operated continuously for several days without column damage. An additional advantage of the proposed tapering approach is that application of pressure on the in- and outlet vial during separation was not required to prevent bubble formation.     

Organized monolayer of thermosensitive microgel beads prepared by double-template polymerization

A 2D close-packed array of thermosensitive microgel beads was prepared by the double-template polymerization method. First, a 2D colloidal crystal of silica beads with 10 microm diameter was obtained by the solvent evaporation method. This monolayer of colloidal crystals can serve as the first template for the preparation of macroporous polystyrene. The macroporous polystyrene trapping the crystalline order can be used as a negative template for fabricating gel beads arrays. A functional surface using thermosensitive poly(N-isopropylacrylamide) gel beads array was fabricated by the double-template polymerization method.     

Integration of the cleanup process: pretreatment of polycyclic aromatic hydrocarbons from diesel exhaust particles on silica gel beads in a microchannel.

This paper presents an integrated microfluidic system that performs cleanup for polycyclic aromatic hydrocarbons (PAHs) from diesel exhaust particles on silica gel beads in a microchip. A column chromatography phase was constructed by filling the silica gel beads into a microchannel that had a dam structure 25 microm high. The height of the dam structure was determined according to the rate of the wet etching. This work on the cleanup of PAHs from diesel exhaust particles showed that the microchip-based system has the same performance as the conventional method on the solid phase extraction column and has some advantages, such as less reagent consumption and shorter pretreatment time, over the conventional method.     

Determination of Diclofenac in Urine Samples by Molecularly‐Imprinted Solid‐Phase Extraction and Adsorptive Differential Pulse Voltammetry

A molecularly imprinted polymer for diclofenac (DCF) was prepared by thermal polymerization over silica beads using 2‐(dimethylamino)ethyl‐methacrylate as functional monomer. After silica elimination by HF treatment, the polymer was applied to the selective solid‐phase extraction of the drug from urine followed by its quantification by adsorptive differential pulse voltammetry. Results indicate that the drug could be selectively extracted from the sample and quantified at clinically relevant concentrations (μg/mL).     

A novel microfluidic concept for bioanalysis using freely moving beads trapped in recirculating flows

There are only a few examples in which beads are employed for heterogeneous assays on microfluidic devices, because of the difficulties associated with packing and handling these in etched microstructures. This contribution describes a microfluidic device that allows the capture, preconcentration, and controlled manipulation of small beads (<6 microm) in etched microchannels using fluid flows only. The chips feature planar diverging and converging channel elements connected by a narrow microchannel. Creation of bi-directional liquid movement by opposing electro-osmotic and pressure-driven flows can lead to the generation of controlled recirculating flow at these elements. Small polymer beads can actually be captured in the controlled rotating flow patterns. The clusters of freely moving beads that result can be perfused sequentially with different solutions. A preliminary binding curve was determined for the reaction of streptavidin-coated beads and fluorescein-labelled biotin, demonstrating the potential of this bead-handling approach for bioanalysis.     

On-chip solid phase extraction coupled with electrophoresis using modified magnetic microspheres as stationary phase

A poly(dimethylsiloxane)(PDMS)/glass hybrid microchip for on-line solid phase extraction (SPE) and electrophoresis separation has been developed and evaluated. The SPE microchannel was crossed to the electrophoresis microchannel. All the microfluidic channels were etched on the glass substrate. The magnetic microspheres were coated with hydroxyl-terminated poly-dimethylsiloxane (PDMS-OH) serving as extraction phase, which could be conveniently immobilized into the sample pretreatment channel by magnetic field. The PDMS-OH microspheres were mobilized into and out of the pretreatment channel by injection flow. The 0.1 μmol/L solution of fluorescence isothiocyanate (FITC)-labeled phenylalanine (Phe) was electrically injected into the SPE channel and extracted onto the PDMS-OH microspheres bed. The enriched FITC-labeled Phe was electrically eluted by 9 mmol/L sodium acetate containing 10% acetonitrile and electrically driven into the electrophoresis channel and then separated. The preconcentration factor could reach 87.5 after sufficient extraction. A linear preconcentration curve was obtained with the initial FITC-labeled Phe concentration ranging from 6 nmol/L to 300 nmol/L (R ²=0.9922) with 200 s loading time. The detection limit (S/N=3) for the FITC-labeled Phe was 3 nmol/L.     

Rapid purification of cell encapsulated hydrogel beads from oil phase to aqueous phase in a microfluidic device

In this paper, we demonstrate a new type of microfluidic chip that can realize continuous-flow purification of hydrogel beads from a carrier oil into aqueous solution by using a laminar-like oil/water interface. The microfluidic chip is composed by two functional components: (1) a flow-focusing bead generation module that can control size and shape of beads, (2) a bead extraction module capable of purifying hydrogel beads from oil into aqueous solution. This module is featured with large branch channels on one side and small ones on the opposite side. Water is continuously infused into the bead extraction module through the large branch channels, resulting in a laminar-like oil/water interface between the branch junctions. Simulation and experimental data show that the efficiency of oil depletion is determined by the relative flow rates between infused water and carrier oil. By using such a microfluidic device, viable cells (HCT116, colon cancer cell line) can be encapsulated in the hydrogel beads and purified into a cell culture media. Significantly improved cell viability was achieved compared to that observed by conventional bead purification approaches. This facile microfluidic chip could be a promising candidate for sample treatment in lab-on-a-chip applications.     

Patterned self-assembled beads in silicon channels.

A novel technique enabling selective bead trapping in microfluidic devices without the use of physical barriers is presented in this paper. It is a fast, convenient and simple method, involving microcontact printing and self-assembly, that can be applied to silicon, quartz or plastic substrates. In the first step, channels are etched in the substrate. The surface chemistry of the internal walls of the channels is then modified by microcontact printing. The chip is submerged in a bead slurry where beads self-assemble based on surface chemistry and immobilize on the internal walls of the channels. Silicon channels (100 microm wide and 50 microm deep) have been covered with monolayers of streptavidin-, amino- and hydroxy-functionalized microspheres and resulted in good surface coverage of beads on the channel walls. A high-resolution pattern of lines of self-assembled streptavidin beads, as narrow as 5 microm, has also been generated on the bottom of a 500 microm wide and 50 microm deep channel. Flow tests were performed in sealed channels with the different immobilized beads to confirm that the immobilized beads could withstand the forces generated by water flowing in the channels. The presented results indicate that single beads can be precisely positioned within microfluidic devices based on self-assembly which is useful as screening and analysis tools within the field of biochemistry and organic chemistry.     

An integrated solid-phase extraction system for sub-picomolar detection

A microchip structure etched on a glass substrate for packed column solid-phase extraction (SPE) and capillary electrochromatography (CEC) is described. A 200 microm long, octadecylsilane (ODS) packed column was secured using two different approaches: solvent lock or polymer entrapment. The former method was utilized for SPE while the latter approach was applied for CEC. In SPE, the ODS packed chamber gave a detection limit of 70 fM for a nonpolar BODIPY (493/503) dye when concentrated for 3 min at an electroosmotic flow rate of 4.14 nL/min, compared to 30 pM for this detector without the SPE step. SPE beds showed reproducible, linear calibration curves (R(2) = 0.9989) between 1 and 100 pM BODIPY at fixed preconcentration times. Breakthrough curves for the 330 pL (ODS-packed) bed indicated a capacity for BODIPY dye of 8.1 x 10(-14) mmol, or 0.25 mmol dye per liter of bed. The ODS-chamber could also be used to analyze dilute amino acid and peptide solutions. In the CEC format, two neutral dyes (BODIPY and acridine orange) were baseline-separated in an isocratic run with a theoretical plate count of 84 (420 000 plates/m) and a reduced plate height of about 1. A labeled peptide was also analyzed by CEC, using the acidic eluent (84% acetonitrile, and 26% aqueous trifluoroacetic acid (0.05%)) preferred for peptide separations on ODS-coated silica particles.     

A valveless microfluidic device for integrated solid phase extraction and polymerase chain reaction for short tandem repeat (STR) analysis

A valveless microdevice has been developed for the integration of solid phase extraction (SPE) and polymerase chain reaction (PCR) on a single chip for the short tandem repeat (STR) analysis of DNA from a biological sample. The device consists of two domains--a SPE domain filled with silica beads as a solid phase and a PCR domain with an ~500 nL reaction chamber. DNA from buccal swabs was purified and amplified using the integrated device and a full STR profile (16 loci) resulted. The 16 loci Identifiler? multiplex amplification was performed using a non-contact infrared (IR)-mediated PCR system built in-house, after syringe-driven SPE, providing an ~80-fold and 2.2-fold reduction in sample and reagent volumes consumed, respectively, as well as an ~5-fold reduction in the overall analysis time in comparison to conventional analysis. Results indicate that the SPE-PCR system can be used for many applications requiring genetic analysis, and the future addition of microchip electrophoresis (ME) to the system would allow for the complete processing of biological samples for forensic STR analysis on a single microdevice.     

Pinched flow fractionation devices for detection of single nucleotide polymorphisms

We demonstrate a new and flexible microfluidic based method for genotyping single nucleotide polymorphisms (SNPs). The method relies on size separation of selectively hybridized polystyrene microspheres in a microfluidic pinched flow fractionation (PFF) device. The microfluidic PFF devices with 13 mum deep channels were fabricated by thermal nanoimprint lithography (NIL) in a thin film of cyclic-olefin copolymer (mr-I T85) on a silicon wafer substrate, and the channels were sealed by thermal polymer bonding. Streptavidin coated polystyrene microspheres with a mean diameter of 3.09 microm and 5.6 microm were functionalized with biotin-labeled oligonucleotides for the detection of a mutant (Mt) or wild-type (Wt) DNA sequence in the HBB gene, respectively. Hybridization to functionalized beads was performed with fluorescent targets comprising synthetic DNA oligonucleotides or amplified RNA, synthesized using human DNA samples from individuals with point mutations in the HBB gene. Following a stringent wash, the beads were separated in a PFF device and the fluorescent signal from the beads was analyzed. Patients being wildtypes, heterozygotes or mutated respectively for the investigated mutation could reliably be diagnosed in the PFF device. This indicates that the PFF technique can be used for accurate and fast genotyping of SNPs.     

原位聚合阴离子交换型固相萃取（SPE）微柱

原位聚合阴离子交换型固相萃取（SPE）微柱;原位聚合;SPE微柱;分离富集     

Automated and parallel microfluidic DNA extraction with integrated pneumatic microvalves/pumps and reusable open-channel columns

A novel microfluidic DNA extraction protocol based on integrated diaphragm microvalves/pumps and silica-deposited open-channel columns was developed specifically for automated and parallel DNA solid-phase extraction (SPE). The method uses microfluidic chips with a sandwiched structure containing three layers, which are the upper fluidic layer with surface-deposited silica on glass open channels as the extraction phase, the lower actuation layer with valve actuation channels on a glass wafer, and the middle poly(dimethylsiloxane) (PDMS) membrane for reversible bonding of the two glass substrates. These two glass substrates can be reused after thoroughly cleaning and the PDMS membrane can be replaced conveniently, which could effectively decrease the time and cost of chip manufacturing. The normally closed microvalves/pumps were used to automatically control all processes of the on-chip DNA SPE without cross-contamination and leakage, enabling the processing of multiple samples in parallel without changing the microvalve control module. Using the microchip device with integrated microvalves/pumps, automated, programmable, and simultaneous λ-DNA extractions from different samples could be attained, even from complex solutions such as human blood, and the silica-deposited open-channel columns could be reused stably and reliably. Results have demonstrated that most of the eluted λ-DNA was recovered in the second 2 µL of elution buffer with high-purity suitable for successful polymerase chain reaction amplification, making it possible for further integration into microfluidic devices for fully functional and high-throughput genetic analysis.     

Uniform-sized molecularly imprinted polymer for metsulfuron-methyl by one-step swelling and polymerization method

Uniform-sized molecularly imprinted polymer (MIP) beads for metsulfuron-methyl (MSM) were firstly prepared by one-step swelling and polymerization method using 4-vinylpyridine (4-VPY) and ethylene glycol dimethacrylate (EDMA) as functional monomer and cross-linker, respectively. The preparation was optimized by varying the ratio of MSM to 4-VPY. The chromatographic behaviors of MSM and other structurally related sulfonylureas (SUs) on the resultant MIP column were evaluated. The imprinted polymer revealed specific affinity to the template and the fair resolution of SUs was also obtained. Furthermore, the uniform-sized MSM-MIP was used as the solid phase extraction (SPE) material to enrich MSM in real water samples before reversed-phase HPLC (RP-HPLC) analysis. The recovery of MSM from 100 mL of drinking water at a 50 ng/L spike level was 99.59% with R.S.D. of 1.13%. The detection limit was about 6.0 ng/L of MSM when enriching a 100 mL water sample.