Power-free sequential injection for microchip immunoassay toward point-of-care testing

This paper presents a simple fluid handling technique for microchip immunoassay. Necessary solutions were sequentially injected into a microchannel by air-evacuated poly(dimethylsiloxane), and were passively regulated by capillary force at the inlet opening. For heterogeneous immunoassay, microchips are potentially useful for reduction of sample consumption and assay time. However, most of the previously reported microchips have limitations in their use because of the needs for external power sources for fluid handling. In this paper, an on-chip heterogeneous immunofluorescence assay without such an external power source is demonstrated. The microchip consisting of poly(dimethylsiloxane) (PDMS) and glass has a simple structure, and therefore is suitable for single-use applications. Necessary solutions were sequentially injected into a microchannel in an autonomous fashion with the power-free pumping technique, which exploits the high solubility and the rapid diffusion of air in PDMS. For deionized water, this method yielded flow rates of 3-5 nL s-1 with reproducibility of 4-10%. The inlet opening of the microchannel functioned as a passive valve to hold the solution when the flow was finished. Rabbit immunoglobulin G (rIgG) and human C-reactive protein (CRP) were detected using the microchannel walls as reaction sites. With the sample consumption of 1 microL and the assay time of approximately 20 min including the antibody immobilization step, the sandwich immunoassay methods for rIgG and CRP exhibited the limits of detection of 0.21 nM (0.21 fmol) and 0.42 nM (0.42 fmol), respectively.     

Electrochemical detector for microchip electrophoresis of poly(dimethylsiloxane) with a three-dimensional adjustor

This paper presents an electrochemical detector for poly(dimethylsiloxane) (PDMS) microchip CE with a three-dimensional adjustor which makes it possible to accurately align a separate working electrode that can be easily fabricated in laboratory to the uncertain PDMS microchannel outlet. The substantial influence of the electrode-PDMS microchannel distance was investigated. The optimal electrode-outlet distance was found to be 10 microm for the PDMS microchannel with the width of 50 microm due to its relatively slow electroosmotic flow. Adrenaline and catechol were well separated, with a linear response range from 20 microM to 1 mM, and a detection limit of 2 microM for catechol, using carbon disk electrode (diameter of 300 microm). Furthermore, arginine and histidine can be well separated and detected directly in the PDMS microchannel using a Cu disk electrode (diameter of 150 microm).     

Development of a sensitive methodology for the analysis of chlorobenzenes in air by combination of solid-phase extraction and headspace solid-phase microextraction

In this study, a combination of solid-phase extraction (SPE) and solid-phase microextraction (SPME) has been used to determine chlorobenzenes in air. Analytes were sampled by pumping a known volume of air through a porous polymer (Tenax TA). Then, the adsorbent was transferred into a glass vial and SPME was performed. The quantification was carried out using gas chromatography (GC)-electron-capture detection or GC-MS. Several SPME coatings (100 microm poly(dimethylsiloxane) (PDMS), 75 microm Carboxen (CAR)-PDMS, 65 microm PDMS-divinylbenzene (DVB), 65 microm PDMS-DVB and 85 microm polyacrylate (PA) were evaluated, obtaining the highest responses with Carbowax (CW)- PDMS for the most volatile chlorobenzenes, and with PDMS-DVB or CW-DVB fibers for the semivolatile compounds. To optimize some other factors that could affect the SPME step, a factorial design was used. Kinetic studies of the SPME process were also performed. Concerning the SPE step, breakthrough was studied, showing that 2.5 m3 of air could be processed without losses of the most volatile compounds. The performance of the method was evaluated. External calibration, which does not require the complete sampling process, demonstrated to be suitable, obtaining good linearity (R2 > 0.99) for all chlorobenzenes. Recovery studies were performed at two concentration levels (4 and 40 ng/m3), obtaining quantitative recoveries (>80%). Limits of detection at the sub ng/m3 were achieved for all the target compounds.     

Separation of three water-soluble vitamins by poly(dimethylsiloxane) microchannel electrophoresis with electrochemical detection

A method for rapid separation and sensitive determination of three water-soluble vitamins, pyridoxine, ascorbic acid (VC), and p-aminobenzoic acid (PABA) has been developed by PDMS microchannel electrophoresis integrated with amperometric detection. After treatment of the microchip with oxygen plasma, the peak shapes of the three analytes were essentially improved. Pyridoxine, VC, and PABA were well separated within only 80 s in a running buffer of 20 mM borate solution (pH 8.5). Good linearity was obtained within the concentration range of 2-200 microM for the three water-soluble vitamins. The detection limits were 1.0 microM for pyridoxine and VC, and 1.5 microM for PABA. The proposed method has been successfully applied to real human urine sample, without solid phase extraction, with recoveries of 80-122% for the three water-soluble vitamins.     

Modular approach to fabrication of three-dimensional microchannel systems in PDMS-application to sheath flow microchips

A modular approach to fabrication of three-dimensional microchannel systems in polydimethylsiloxane (PDMS) is presented. It is based on building blocks with microstructuring on up to three faces. The assembled 3D-microchip consists of three building blocks in two layers. For assembly of the bottom layer two building blocks are joined horizontally, whereby the side structuring of the first is sealed against the flat side surface of the other. This results in the formation of a vertical interconnection opening between the building blocks to supplement the microstructuring on the lower faces. The 3D microchannel system is completed by placing a third building block, with microstructuring only on its lower face, on top of the assembled layer. While plasma assisted bonding is used between the two building blocks of the bottom layer, inherent adhesion is sufficient between the layers and for attaching the assembled 3D-microchip to a substrate. This modular approach was applied to the fabrication of a 3D-sheath flow microchip. It comprises a 20 microm deep microchannel system with sample inlet, open sensing area and outlet in the bottom layer and sheath flow inlet in the top layer. 100 microM fluorescein at 6 microL min(-1) was used as sample flow and water at increasing flow rates as sheath flow. With ratios of sheath to sample flow up to 20:1 sample layers down to 1 microm thickness could be generated. Sample layer thickness was determined via volume detection on an epi-fluorescence microscope followed by image analysis.     

Covalent modified hydrophilic polymer brushes onto poly(dimethylsiloxane) microchannel surface for electrophoresis separation of amino acids

A new environmentally friendly method is developed for preventing nonspecific biomolecules from adsorption on poly(dimethylsiloxane) (PDMS) surface via in situ covalent modification. o-[(N-Succinimdyl)succiny]-o'-methyl-poly(ethylene glycol) (NSS-mPEG) was covalently grafted onto PDMS microchannel surface that was pretreated by air-plasma and silanized with 3-aminopropyl-triethoxysilanes (APTES). The modification processes were carried out in aqueous solution without any organic solvent. The mPEG side chains displayed extended structure and created a nonionic hydrophilic polymer brushes layer on PDMS surface, which can effectively prevent the adsorption of biomolecules. The developed method had improved reproducibility of separation and stability of electroosmotic flow (EOF), enhanced hydrophilicity of surface and peak resolution, and decreased adsorption of biomolecules. EOF in the modified microchannel was strongly suppressed, compared with those in the native and silanized PDMS microchips. Seven amino acids have been efficiently separated and successfully detected on the coated PDMS microchip coupled with end-channel amperometric detection. Relative standard deviations (RSDs) of their migration time for run-to-run, day-to-day and chip-to-chip, were all below 2.3%. Moreover, the covalent-modified PDMS channels displayed long-term stability for 4 weeks. This novel coating strategy showed promising application in biomolecules separation.     

Simultaneous analysis of polychlorinated biphenyls and organochlorine pesticides in water by headspace solid-phase microextraction with gas chromatography-tandem mass spectrometry

Headspace solid-phase microextraction combined with gas chromatography-ion trap tandem mass spectrometry (HS-SPME-GC-ITMS-MS) method has been developed and studied for the simultaneous determination of 15 organochlorine pesticides (OCPs) and 20 polychlorinated biphenyls (PCBs) in aqueous samples. To perform the HS-SPME polydimethylsiloxane (PDMS) (7, 30 and 100 microm film thickness) and polydimethylsiloxane-divinylbenzene (PDMS-DVB) fibers were initially compared on the basis of their absorption capacities for the selected compounds, and PDMS 100 microm film thickness was selected to accomplish the rests of essays. The influence of various parameters on OCPs and PCBs extraction efficiency by HS-SPME was thoroughly studied using GC-electron capture detector (ECD). Parameters such as collision induced dissociation (CID) resonant excitation amplitude and RF storage level were optimized to increase specificity and sensibility for ITMS-MS analysis. The performance of proposed HS-SPME-GC-ITMS-MS methodology with respect to linearity, reproducibility and limit of detection (LOD) was evaluated by water spiked with target compounds. The linear range of most compounds was found to be between 0.01 and 1 ng mL(-1) and the limits of detection were between 0.4 and 26 pg mL(-1). The reproducibility of the method (n = 6), expressed as relative standard deviation (RSD), was between 5 and 21%. Finally, developed procedure was applied to determine selected OCPs and PCBs in river water samples in concentration below 0.1 ng mL(-1) can be easily carried out with ultra-selectivity and precision.     

Oxygen consumption of cell suspension in a poly(dimethylsiloxane) (PDMS) microchannel estimated by scanning electrochemical microscopy

A quantitative analysis of the oxygen concentration profile near a poly(dimethylsiloxane) (PDMS) microfluidic device was performed using scanning electrochemical microscopy (SECM). A microchannel filled with sodium sulfite (Na(2)SO(3)) aqueous solution was imaged by SECM, showing that the oxygen diffusion layer of the PDMS microchannel was observed to be hemicylindrical. Based on a theoretical analysis of the hemicylindrical diffusion layer of the microchannel, the total oxygen mass transfer rates of oxygen to the PDMS microchannel filled with the Na(2)SO(3) solution was calculated to be (4.01 +/- 0.30) x 10(-12) mol s(-1). This is the maximum value of the oxygen transfer rate for this PDMS microchannel device. The oxygen consumption rate increased almost linearly with the logarithm of the concentration of E. coli cells (10(6) approximately 10(8) cells). The respiratory activity for a single E. coli cell was estimated to be approximately 4.31 x 10(-20) mol s(-1) cell(-1).     

Continuous hydrophoretic separation and sizing of microparticles using slanted obstacles in a microchannel

We report a microfluidic separation and sizing method of microparticles with hydrophoresis--the movement of suspended particles under the influence of a microstructure-induced pressure field. By exploiting slanted obstacles in a microchannel, we can generate a lateral pressure gradient so that microparticles can be deflected and arranged along the lateral flows induced by the gradient. Using such movements of particles, we completely separated polystyrene microbeads with 9 and 12 microm diameters. Also, we discriminated polystyrene microbeads with diameter differences of approximately 7.3%. Additionally, we measured the diameter of 10.4 microm beads with high coefficient of variation and compared the result with a conventional laser diffraction method. The slanted obstacle as a microfluidic control element in a microchannel is analogous to the electric, magnetic, optical, or acoustic counterparts in that their function is to generate a field gradient. Since our method is based on intrinsic pressure fields, we could eliminate the need for external potential fields to induce the movement of particles. Therefore, our hydrophoretic method will offer a new opportunity for power-free and biocompatible particle control within integrated microfluidic devices.     

Rapid fabrication of microfluidic devices in poly(dimethylsiloxane) by photocopying

A very simple and fast method for the fabrication of poly(dimethylsiloxane) (PDMS) microfluidic devices is introduced. By using a photocopying machine to make a master on transparency instead of using lithographic equipment and photoresist, the fabrication process is greatly simplified and speeded up, requiring less than 1.5 h from design to device. Through SEM characterization, any micro-channel network with a width greater than 50 microm and a depth in the range of 8-14 microm can be made by this method. After sealing to a Pyrex glass plate with micromachined platinum electrodes, a microfluidic device was made and the device was tested in FIA mode with on-chip conductometric detection without using either high voltage or other pumping methods.     

Deoxyribonucleic acid modified poly(dimethylsiloxane) microfluidic channels for the enhancement of microchip electrophoresis

In this paper, deoxyribonucleic acid (DNA) was employed to construct a functional film on the PDMS microfluidic channel surface and apply to perform electrophoresis coupled with electrochemical detection. The functional film was formed by sequentially immobilizing chitosan and DNA to the PDMS microfluidic channel surface using the layer-by-layer assembly. The polysaccharide backbone of chitosan can be strongly adsorbed onto the hydrophobic PDMS surface through electrostatic interaction in the acidic media, meanwhile, chitosan contains one protonatable functional moiety resulting in a strong electrostatic interactions between the surface amine group of chitosan and the charged phosphate backbone of DNA at low pH, which generates a hydrophilic microchannel surface and reveals perfect resistance to nonspecific adsorption of analytes. Aminophenol isomers (p-, o-, and m-aminophenol) served as a separation model to evaluate the effect of the functional PDMS microfluidic chips. The results clearly showed that these analytes were efficiently separated within 60 s in a 3.7 cm long separation channel and successfully detected on the modified microchip coupled with in-channel amperometric detection mode at a single carbon fiber electrode. The theoretical plate numbers were 74,021, 92,658 and 60,552 N m^?1 at the separation voltage of 900 V with the detection limits of 1.6, 4.7 and 2.5 μM (S/N = 3) for p-, o-, and m-aminophenol, respectively. In addition, this report offered an effective means for preparing hydrophilic and biocompatible PDMS microchannel surface, which would facilitate the use of microfluidic devices for more widespread applications.     

Development of a rapid and sensitive method for the simultaneous determination of 1,2-dibromoethane, 1,4-dichlorobenzene and naphthalene residues in honey using HS-SPME coupled with GC-MS

A new method for the simultaneous determination of 1,4-dichlorobenzene (p-DCB), naphthalene and 1,2-dibromoethane (1,2-DBE) residues in honey has been developed. Analysis is carried out using gas chromatography-mass spectrometry (GC/MS) in selected ion monitoring mode (SIM), after extraction and preconcentration of target analytes by headspace solid-phase microextraction (HS-SPME), with a 100 microm film thickness polydimethylsiloxane (PDMS) fiber. Several parameters affecting the extension of the adsorption process (i.e., addition of salt, extraction time, extraction temperature) were studied. The optimal conditions for the determination of these analytes were established. The proposed HS-SPME method showed good sensitivity, without carryover between the samples. Linearity was studied from 5 to 2500 microg kg(-1) for p-DCB, 0.5 to 500 microg kg(-1) for naphthalene and 5 to 500 microg kg(-1) honey for 1,2-DBE with correlation coefficients (r(2)) ranging from 0.9901 to 0.9999. Precision was assessed and both intra and inter-day R.S.D.s (%) were below 6.3%. The detection limits were found to be 1, 0.1 and 2 microg kg(-1) honey for p-DCB, naphthalene and 1,2-DBE, respectively. The percentage recoveries that were evaluated with the proposed HS-SPME method and the standard addition calibration technique gave values among 72.8 and 104.3% for measurements in samples spiked with one target analyte or mixtures of the three. This method has been applied for the analysis of unknown honey samples. The results showed an excellent applicability of the proposed method for the determination of the target compounds in honey samples.     

Laser-induced cavitation based micropump

Lab-on-a-chip devices are in strong demand as versatile and robust pumping techniques. Here, we present a cavitation based technique, which is able to pump a volume of 4000 microm3 within 75 micros against an estimated pressure head of 3 bar. The single cavitation event is created by focusing a laser pulse in a conventional PDMS microfluidic chip close to the channel opening. High-speed photography at 1 million frames s(-1) resolves the flow in the supply channel, pump channel, and close to the cavity. The elasticity of the material affects the overall fluid flow. Continuous pumping at repetition rates of up to 5 Hz through 6 mm long square channels of 20 microm width is shown. A parameter study reveals the key-parameters for operation: the distance between the laser focus and the channel, the maximum bubble size, and the chamber geometry.     

Immunosensing of Staphylococcus enterotoxin B (SEB) in milk with PDMS microfluidic systems using reinforced supported bilayer membranes (r-SBMs)

A versatile and novel method has been developed for microfluidic immunosensing of the food-borne pathogen Staphylococcus enterotoxin B (SEB) in poly(dimethylsiloxane) (PDMS) chips. Supported bilayer membranes (SBMs) were generated by vesicle fusion in oxidized PDMS microchannels for minimizing non-specific adsorption of biomolecules. The stability of SBMs was strengthened with a streptavidin layer to make them air-stable and allow for subsequent display of the biotin-functionalized antibodies. The reinforced supported bilayer membranes (r-SBMs) are fluid, exhibiting a lateral diffusion coefficient of approximately 1.9 microm(2) s(-1), and no detectable change of mobility was found after dehydration/rehydration. This is a substantial improvement over phosphatidylcholine (PC) membranes on PDMS, which suffered a roughly 10% reduction in the mobile fraction and 30% decrease in mobility after dehydration. Non-specific protein adsorption in the membrane-treated channels was reduced 100-1000 fold as compared to PDMS surfaces without a membrane coating. A flow-based microfluidic immunosensor for SEB was developed using antibodies linked to the r-SBMs in PDMS channels, and a detection limit of 0.5 ng mL(-1) was obtained from the linear portion of the calibration curve. The microchip was applied to detection of SEB in milk, and similar response and sensitivity were obtained, demonstrating the sensor's remarkable performance for real world samples. The r-SBMs overcome the stability hurdle in SBM-modified surfaces, opening up possibilities for transport and storage of membrane-functionalized microchips in the dehydrated form without compromising the performance, and facilitating the commercialization of disposable SBM-based microdevices.     

Rapid microRNA detection using power-free microfluidic chip: coaxial stacking effect enhances the sandwich hybridization

We present a new method for rapid microRNA detection with a small volume of sample using the power-free microfluidic device driven by degassed PDMS. Target microRNA was detected by sandwich hybridization taking advantage of the coaxial stacking effect. This method allows us to detect miR-21 in 20 min with a 0.5 μL sample volume at a limit of detection of 0.62 nM. Since microRNAs can act as cancer markers, this method might substantially contribute to future point-of-care cancer diagnosis.     

Fabrication of carbon microelectrodes with a micromolding technique and their use in microchip-based flow analyses

In this paper, we report a new technique to pattern carbon microelectrodes for use in microfluidics. This technique, termed micromolding of carbon inks, uses poly(dimethylsiloxane)(PDMS) microchannels to define the size of the microelectrode. First, PDMS microchannels of the approximate dimensions desired for the microelectrode are made by soft lithography. The PDMS is then reversibly sealed to a substrate and the microchannels are filled with carbon ink. After a heating step the PDMS mold is removed, leaving a carbon microelectrode with a size slightly smaller than the original PDMS microchannel. The resulting microelectrode (27 microm wide and 6 microm in height) can be reversibly sealed to a PDMS-based flow channel. Fluorescence microscopy showed that no leakage occurred around the chip/electrode seal, even up to flow rates of 10 microL min(-1). The electrode was characterized by microchip-based flow injection analysis. Injections of catechol in Hank's Balanced Salt Solution (pH 7.4), showed a linear response from 2 mM to 10 microM (r(2)= 0.995), with a sensitivity of 56.5 pA microM(-1) and an estimated limit of detection of 2 microM (0.27 picomole, S/N=3). Reproducibility of the electrode response was shown by repeated injections (n= 10) of a 500 microM catechol solution, resulting in a RSD of 4.6%. Finally, selectivity was demonstrated by coating the microelectrode with Nafion, a perfluoronated cation exchange polymer. Dopamine exhibited a response at the modified microelectrode while ascorbic acid was rejected by the Nafion-coating. These electrodes provide inexpensive detectors for microfluidic applications while also being viable alternatives to use of other carbon microelectrode materials, such as carbon fibers. Furthermore, the manner in which the microelectrodes are produced will be of interest to researchers who do not have access to state of the art microfabrication facilities.     

Determination of phthalates in water using fiber introduction mass spectrometry

Fiber introduction mass spectrometry (FIMS)-a direct coupling of SPME and MS-using selective ion monitoring (SIM) was used to detect and quantify dimethylphthalate (DMP), diethylphthalate (DEP) and dipropylphthalate (DPP) in mineral water. In FIMS, a chromatographic silicone septum is the only barrier between ambient and the high-vacuum mass spectrometer, permitting direct introduction of the SPME fiber into the ionization region of the equipment. After their thermal desorption and ionization and dissociation, the extracted phthalates are detected and quantitated by MS. Three types of SPME fibers were screened for best analyte sorption/desorption behaviors: 100 microm polydimethylsiloxane (PDMS), 65 microm polydimethylsiloxane/divinylbenzene (PDMS/DVB) and 65 microm Carbowax/divinylbenzene (CW/DVB). The PDMS/DVB and CW/DVB fibers were then evaluated for precision, and quantitative figures of merit were assessed for extractions using the PDMS/DVB fiber, which displayed the best overall performance. FIMS with the PDMS/DVB fiber allows simple extraction and MS detection and quantitation of DMP in water with good linearity and precision, and at concentrations as low as 3.6 microg L(-1). The LD and LQ of FIMS are below the maximum phthalate concentration allowed by the USEPA for drinking water (6 microg L(-1)).     

Design of a gold nanoprobe for rapid and portable mercury detection with the naked eye

A gold nanoprobe that can respond colorimetrically to Hg(2+) is designed and coupled with a power-free PDMS device; the system can be used for rapid and visual detection of low micromolar Hg(2+) in real environmental samples.     

Velocity measurement of particulate flow in microfluidic channels using single point confocal fluorescence detection.

This article presents a non-invasive, optical technique for measuring particulate flow within microfluidic channels. Confocal fluorescence detection is used to probe single fluorescently labeled microspheres (0.93 microm diameter) passing through a focused laser beam at a variety of flow rates (50 nL min(-1)-8 microL min(-1)). Simple statistical methods are subsequently used to investigate the resulting fluorescence bursts and generate velocity data for the flowing particles. Fluid manipulation is achieved by hydrodynamically pumping fluid through microchannels (150 microm wide and 50 microm deep) structured in a polydimethylsiloxane (PDMS) substrate. The mean fluorescence burst frequency is shown to be directly proportional to flow speed. Furthermore, the Poisson recurrence time and width of recovered autocorrelation curves is demonstrated to be inversely proportional to flow speed. The component-based confocal fluorescence detection system is simple and can be applied to a diversity of planar chip systems. In addition, velocity measurement only involves interrogation of the fluidic system at a single point along the flow stream, as opposed to more normal multiple-point measurements.     

A passive pumping method for microfluidic devices

The surface energy present in a small drop of liquid is used to pump the liquid through a microchannel. The flow rate is determined by the volume of the drop present on the pumping port of the microchannel. A flow rate of 1.25 microL s(-1) is demonstrated using 0.5 microL drops of water. Two other fluid manipulations are demonstrated using the passive pumping method: pumping liquid to a higher gravitational potential energy and creating a plug within a microchannel.