Continuous flow microfluidic device for cell separation, cell lysis and DNA purification

A novel integrated microfluidic device that consisted of microfilter, micromixer, micropillar array, microweir, microchannel, microchamber, and porous matrix was developed to perform sample pre-treatment of whole blood. Cell separation, cell lysis and DNA purification were performed in this miniaturized device during a continuous flow process. Crossflow filtration was proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells were lyzed in guanidine buffer, genomic DNA in white blood cells was released and adsorbed on porous matrix fabricated by anodizing silicon in HF/ethanol electrolyte. The flow process of solutions was simulated and optimized. The anodization process of porous matrix was also studied. Using the continuous flow procedure of cell separation, cell lysis and DNA adsorption, average 35.7 ng genomic DNA was purified on the integrated microfluidic device from 1 μL rat whole blood. Comparison with a commercial centrifuge method, the miniaturized device can extract comparable amounts of PCR-amplifiable DNA in 50 min. The greatest potential of this integrated miniaturized device was illustrated by pre-treating whole blood sample, where eventual integration of sample preparation, PCR, and separation on a single device could potentially enable complete detection in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

Determination of trace and major elemental profiles in street dust samples by fast miniaturized ultrasonic probe extraction and ICP-MS

A simple, fast and miniaturized ultrasonic probe assisted protocol for acid extraction of trace and major elements from street dust samples, prior to final determination by ICP-MS, has been optimized and validated by standard reference materials and applied during an urban air quality monitoring campaign. 15 mg samples were treated for 3 min with 1 mL concentrated HNO₃-HCl (1:3, v/v) extracting solution, by a 1 mm diameter titanium sonotrode connected to a 200 W and 24 kHz ultrasonic device at 80% amplitude. After centrifugation, the extracts were assayed by ICP-MS with good recoveries for the certified elements. Statistical analysis of real sample results by cluster analysis allowed the correct grouping of the samples according to the influence of traffic and construction/demolition activities. Correlation of street dust and PM10 elemental profiles showed the potential applicability of the proposed analytical protocol as a simple and effective way for urban air quality monitoring.     

Design and development of microfabricated capillary electrophoresis devices with electrochemical detection

Our efforts have been focused on developing a self-contained and transportable microfabricated electrophoresis (CE) system with integrated electrochemical detection (ED). The current prototype includes all necessary electrodes “on-chip” and utilizes miniaturized CE and ED supporting electronics custom designed for this purpose. State-of-the-art design/modeling tools and novel microfabrication procedures were used to create recessed platinum electrodes with complex geometries and the CE/ED device from two patterned ultra-flat glass substrates. The electrodes in the bottom substrate were formed by a self-aligned etch and deposition technique followed by a photolithographic lift-off process. The microchannels (20 μm deep×65 μm wide (average)) were chemically etched into the top substrate followed by thermal bonding to complete the microchip device. CE/ED experiments were performed using 0.02 M phosphate buffer (pH 6), an analyte/buffer solution (2.2 mM dopamine, 2.3 mM catechol) and varying separation voltages (0-500 V) with a custom electronics unit interfaced to a laptop computer for data acquisition. Detection limits (S/N=3) were found to be at the micromolar level and a linear detection response was observed up to millimolar concentrations for dopamine and catechol. The microchip CE/ED system injected 50 pl volumes of sample, which corresponded to mass detection limits on the order of 200 amol. For the first time, an integrated “on-chip” multi-electrode array CE/ED device was successfully designed, fabricated and tested. The majority of the electrodes (six out of eight) in the array were capable of detecting dopamine with the amplitude of the signal (i.e., peak heights) decreasing as the electrode distance from the channel exit increased.     

A cold plasma dielectric barrier discharge atomic emission detector for atmospheric mercury

An automated atmospheric elemental mercury analyzer based on the dielectric barrier discharge (DBD) atomic emission technique was developed. The instrument is based on a gold-on tungsten coiled filament preconcentrator fashioned from commercial quartz-halogen lamps, a DBD excitation source and a radiation detector. An in-house program provided system control and data collection. Several types of radiation detectors, e.g., charge coupled device (CCD) array spectrometers, photomultiplier tubes (PMTs) and phototube (PT) are investigated. An argon plasma provided better performance than a nitrogen plasma. With ∼0.88 standard liters per min sampling rate and preconcentration for 2 min, the estimated (S/N = 3) detection limit was 0.12 ng/L (Hg⁰), the linear range extended at least to 6.6 ng Hg/L. Typical RSD values for determination at the single digit ng/L level ranged from 2.8 to 4.9%. In 19 separate calibrations conducted over 7 days, the calibration slope had a standard error of 1%. The system was applied to the determination of atmospheric mercury in two different locations.     

Rapid chemiluminescence biosensing of microcystin-LR

A rapid immunoassay for sensitive detection of microcystin-LR using a portable chemiluminescence multichannel immunosensor (CL-MADAG) was developed. The sensor device is based on a capillary ELISA technique in combination with a miniaturized fluidics system and uses chemiluminescence as the detection principle. Minimum concentrations of at least 0.2 μg L⁻¹ microcystin-LR could be unambiguously measured in a spiked buffer system as well as in spiked real water samples. A single sample analysis for detection of microcystin-LR could be accomplished in just 13 min on the CL-MADAG. Besides providing a highly reproducible, fast and easy to perform test format, one major advantage of the newly established capillary immunoassay is represented by the feasibility of an internal retrospective quality control mechanism. Finally, simultaneous CL-MADAG measurements employing our inhibition immunoassay and a sandwich ELISA could be successfully demonstrated.     

Novel membrane-based sensor for online membrane integrity monitoring

A novel membrane-based sensor device for upstream membrane integrity monitoring has been developed and evaluated in this study. The sensor is based on relative trans-membrane pressures created by two membranes in series inside the sensor device that detects deposition from the sample stream onto the first of the sensor membranes. The sensor pressure signals can distinguish between intact or damaged membranes in the upstream membrane filtration process. Studies were conducted to evaluate both stabilities and sensitivities of the relative trans-membrane pressure monitoring technique. Sensitivity, based on the response times of the membrane sensor for particle detection, was determined for a range of operating conditions, membrane sandwich configurations, and particle concentrations in both simulated membrane failures and for actual pin-hole defects on a submerged MF membrane. The results showed that both sensitivities and stability strongly depended on membrane sandwich configurations (membrane characteristics) in the sensor, and mode of operation (pressurized or vacuum). The membrane sensor detected bentonite particles with a concentration of 0.3 mg/L (turbidity ∼0.3 NTU) in approximately 35 min in the vacuum mode. The sensor is reliable, sensitive and low cost. It has potential applications in decentralized systems or in multichannel monitoring of local conditions in a large plant. Possible applications of the membrane sensor for fouling monitoring are also discussed.     

Post-column detection of benzenediols and 1,2,4-benzenetriol based on acidic potassium permanganate chemiluminescence

Based on the sensitizing effect of formic acid on the chemiluminescence (CL) reaction of polyhydroxylbenzenes with acidified potassium permanganate and the combination technique of high-performance liquid chromatography (HPLC), a sensitive, selective and simple post-column CL detection method for simultaneously determining catechol, resorcinol, hydroquinone and 1,2,4-benzenetriol is described. The optimal conditions for the CL detection and HPLC separation were carried out. The linear ranges were: 6.0 × 10⁻³-1.5 mg/L for hydroquinone, 8.0 × 10⁻³-1.5 mg/L for 1,2,4-benzenetriol, 1.0 × 10⁻²-2.0 mg/L for resorcinol and 1.0 × 10⁻²-2.5 mg/L for catechol, respectively. The detection limits are: 3.2 × 10⁻³ mg/L for hydroquinone, 3.9 × 10⁻³ mg/L for 1,2,4-benzenetriol, 4.7 × 10⁻³ mg/L for resorcinol and 5.2 × 10⁻³ mg/L for catechol, respectively. Combining with solid phase extraction, the proposed method has been successfully applied to the determination of the polyhydroxylbenzenes in river water. The recoveries for three benzentriols were 92.1-95.4% and 82.0% for 1,2,4-benzenetriol, respectively.     

Electrocatalytic detection of insulin at RuOx/carbon nanotube-modified carbon electrodes

A bilayer surface coating, prepared by electrodepositing ruthenium oxide (RuOx) onto a carbon nanotube (CNT) layer, offers dramatic improvements in the stability and sensitivity of voltammetric and amperometric measurements of insulin compared to the individual (CNT or RuOx) coated electrodes. The enhanced electrocatalytic activity towards insulin is indicated from lowering the potential of the oxidation process (starting around 0.35 versus Ag/AgCl) and the substantially higher sensitivity over the entire potential range. A wide linear dynamic range (10-800 nM) was achieved with a detection limit of 1 nM. The marked electrocatalytic activity of the RuOx/CNT coating towards insulin is coupled with a greatly enhanced stability. For example, the insulin amperometric response of the RuOx/CNT-coated electrodes is highly stable, with 97% of the initial activity remaining after 60 min stirring of 2 × 10⁻⁶ M solution (compared to significantly faster current diminutions at the RuOx- or CNT-coated surfaces). The results suggest great promise for miniaturized sensors and detectors for monitoring insulin.     

Graphene nano-ink biosensor arrays on a microfluidic paper for multiplexed detection of metabolites

The development of a miniaturized and low-cost platform for the highly sensitive, selective and rapid detection of multiplexed metabolites is of great interest for healthcare, pharmaceuticals, food science, and environmental monitoring. Graphene is a delicate single-layer, two-dimensional network of carbon atoms with extraordinary electrical sensing capability. Microfluidic paper with printing technique is a low cost matrix. Here, we demonstrated the development of graphene-ink based biosensor arrays on a microfluidic paper for the multiplexed detection of different metabolites, such as glucose, lactate, xanthine and cholesterol. Our results show that the graphene biosensor arrays can detect multiple metabolites on a microfluidic paper sensitively, rapidly and simultaneously. The device exhibits a fast measuring time of less than 2 min, a low detection limit of 0.3 μM, and a dynamic detection range of 0.3–15 μM. The process is simple and inexpensive to operate and requires a low consumption of sample volume. We anticipate that these results could open exciting opportunities for a variety of applications.     

Hand-held amperometric sensor for saliva and other oral fluid-based diagnostics

A new hand-held amperometric sensor for monitoring of saliva and other oral fluid-based components is described. The amperometric sensor includes a flow through amperometric detector coupled with a micropipette, and an electronic block which consists of an amplifier, peak detector, microprocessor and display. All operations, such as sample injection, electrochemical detection and data processing are accomplished using only one action, by pressing the plunger of the micropipette. The instrument has been applied for assay of salivary peroxidase, human luteinizing hormone and human chorionic gonadotropin. The low detection limit of peroxidase is 0.5 ng/ml (defined as the concentration which gives a signal with S/N>2). The response time of the sensor is 1-3 s. The linear dynamic range of the hormones is from 1.0 to 80 mUI/ml. The life time of the hand-held amperometric device without regeneration of the electrode surface is 12 months. The sensor has the potential as a tool for noninvasive physiological studies and diagnostic strategies.     

Trace determination of airborne polyfluorinated iodine alkanes using multisorbent thermal desorption/gas chromatography/high resolution mass spectrometry

A novel gas chromatography/high resolution mass spectrometry method coupled with multisorbent thermal desorption cartridges has been developed for the determination of volatile neutral polyfluorinated iodine alkanes (PFIs) in airborne samples. It allows, for the first time, simultaneous analysis of four mono-iodized perfluorinated alkanes, three diiodized perfluorinated alkanes and four mono-iodized polyfluorinated telomers in ambient air samples. 3.75 L air sample was passed through a sorbent tube packed with 150 mg of Tenax TA and 200 mg of Carbograph 1TD for analyte adsorption. Important factors during the analysis procedures, such as safe sampling volume, air sampling rate, analyte desorption and transfer strategies, were optimized and good thermal desorption efficiencies were obtained. The method detection limit (MDL) concentration ranged from 0.04 pg/L for 1H,1H,2H,2H-perfluorododecyl iodide to 1.2 pg/L for perfluorohexyl iodide, and instrument response of a seven-point calibration was linear in the range of 10–1000 pg. Travel spike recoveries ranged from 83% to 116%. Small variabilities of less than 36% were obtained near the MDLs and the differences between triplicates were even smaller (2.1–7.3%) at 200 pg spiked level. The method was successfully applied to analyze ambient air samples collected near a point source, and five PFIs were identified (10.8–85.0 pg/L), with none of the analytes detectable at the background site.     

Implementation of droplet-membrane-droplet liquid-phase microextraction under stagnant conditions for lab-on-a-chip applications

In the current work, droplet-membrane-droplet liquid-phase microextraction (LPME) under totally stagnant conditions was presented for the first time. Subsequently, implementation of this concept on a microchip was demonstrated as a miniaturized, on-line sample preparation method. The performance level of the lab-on-a-chip system with integrated microextraction, capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection in a single miniaturized device was preliminarily investigated and characterized. Extractions under stagnant conditions were performed from 3.5 to 15 μL sample droplets, through a supported liquid membrane (SLM) sustained in the pores of a small piece of a flat polypropylene membrane, and into 3.5-15 μL of acceptor droplet. The basic model analytes pethidine, nortriptyline, methadone, haloperidol, and loperamide were extracted from alkaline sample droplets (pH 12), through 1-octanol as SLM, and into acidified acceptor droplets (pH 2) with recoveries ranging between 13 and 66% after 5 min of operation. For the acidic model analytes Bodipy FL C₅ and Oregon Green 488, the pH conditions were reversed, utilizing an acidic sample droplet and an alkaline acceptor droplet, and 1-octanol as SLM. As a result, recoveries for Bodipy FL C₅ and Oregon Green 488 from human urine were 15 and 25%, respectively.     

A sensitive DNA electrochemical biosensor based on magnetite with a glassy carbon electrode modified by muti-walled carbon nanotubes in polypyrrole

A novel sensitive electrochemical biosensor based on magnetite nanoparticle for monitoring DNA hybridization by using MWNT-COOH/ppy-modified glassy carbon electrode is described. In this new detection system, mercapatoacetic acid (RSH)-coated magnetite nanoparticles, capped with 5′-(NH₂) oligonucleotide, is used as DNA probe to complex 29-base polynucleotide target (a piece of human porphobilinogen deaminase PBGD promoter from 170 to 142). Target sequence hybridized with the probe results in the decrease of the reduction peak current of daunomycin connected with probe. The response of non-complementary sequence was almost the same as the blank, and the response of three-base mismatched sequence within 29-base polynucleotide was obviously distinguished from complementary sequence, which can easily identify point mutation of DNA. The equation of calibration plot is i_p (μA) = 0.8255 − 0.0847c_{target oligonucleotide} × 10¹³ in the range of 6.9 × 10⁻¹⁴ to 8.6 × 10⁻¹³ mol/L, and correlation coefficient is 0.9974. The detective limit is 2.3 × 10⁻¹⁴ mol/L of target oligonucleotide. This device can be optimized for the detection of complex sequence.     

Construction and evaluation of a flow injection micro-analyser based on urethane-acrylate resin

A flow injection micro-analyser with an integrated injection device and photometric detection is described. Channels measuring 205-295 μm depth by 265-290 μm maximum width were manufactured by deep UV lithography on two layers of urethane-acrylate oligomers-based photoresist. Hypodermic syringe needles (450 μm diameter) were connected to the channels for introduction of solutions into the system. Plastic optical fibres were connected to the ends of a 5.0 mm long channel, in order to conduct the light from and to a homemade photometer. The device has a total volume of 7.0 μL and three different sample volumes (0.09, 0.22 and 0.30 μL) can be inserted into the system by choosing the appropriate loop of the hydrodynamic injection approach. The micro-analyser, designed as a single line manifold, was evaluated by determining chloride in waters (mercuric thiocyanate method), and chromium (VI) in wastewater and total chromium in metallic alloys (diphenylcarbazide method). For chloride determination two micro-pumps were employed to impel the solutions, while for chromium determination this task was performed by a conventional peristaltic pump. The results obtained in all determinations did not differ significantly from the reference methods at a confidence level of 95%. In the chloride determination, a flow rate of 50 μL min⁻¹ was used, providing a sample frequency of 45 injection h⁻¹, generating ca. 0.7 mg of Hg(II) after an 8-h working day (ca. 20 mL of solution). This result suggests the potential of the micro-analyser towards the reduction of waste, following the philosophy of Green Chemistry.     

Miniaturized capillary electrophoresis system with ultraviolet photometric detection combined with flow injection sample introduction using a modified falling-drop interface

A miniaturized capillary electrophoresis (CE) system with UV-Vis detection was coupled to a flow injection (FI) system for achieving high throughput continuous sample introduction. The cassette of a commercial CE instrument was modified to hold a 6.5 cm long silica capillary and a flow-through waste reservoir. The cassette was inserted into the flow-cell chamber of a commercial UV detector, with the light beam focused on the capillary and collected by two ball lenses on the cassette. The capillary inlet, left outside the cassette and detector, was positioned on the top of a vertical 3.5 mm diameter glass rod, in close contact with an electrode. Samples injected through the FI system dropped freely on top of the pillar, covering the capillary inlet and electrode. Continuous sample introduction was achieved for CE separations under non-interrupted separation voltage, which was isolated from the FI system through the discontinuity of droplets. The newly developed interface and UV detection system was used for fast separation of sulphamethoxazole (SMZ) and trimethoprim (TMP) in sulphatrim tablets, achieving a high throughput of over 48 h⁻¹, and a low carryover of 2%. Separation efficiencies of 8 μm plate height and detection limits of 1.0 mg l⁻¹ for SMZ and 0.5 mg l⁻¹ (3σ) for TMP were obtained.     

A novel miniaturized homogenous liquid–liquid solvent extraction-high performance liquid chromatographic-fluorescence method for determination of ultra traces of polycyclic aromatic hydrocarbons in sediment samples

A novel technique called miniaturized homogeneous liquid–liquid extraction (MHLLE) followed by high performance liquid chromatographic-fluorescence detection (HPLC-FL) was developed for the extraction and determination of some polycyclic aromatic hydrocarbons (PAHs) as model for analytical problem in sediment samples. The method is based on the rapid extraction of PAHs from a methanolic sample solution into 0.5 mL n-hexane, as a solvent of lower density than water. After addition of water, the extracting solvent immediately forms a distinct water-immiscible phase at the top of the vial, which can be easily separated, evaporated and re-dissolved in 25 μL of methanol and injected to the HPLC instrument. The parameters affecting the extraction process such as type and volume of organic extraction solvent, extraction time, and salt addition were investigated and the partition coefficient between methanol/water–n-hexane phases was evaluated and used to predict the extraction efficiency. Under optimal conditions, the limits of detection were estimated for the individual PAHs as 3S_b (three times of the standard deviation of baseline) of the measured chromatogram, are in the range of 0.003–0.04 ng g⁻¹ for sediment samples. The relative recoveries of PAHs at spiking levels of 1.0 ng g⁻¹ for sediment samples were in the range of 81–92%. The method was also applied to a corresponding standard references materials (IAEA-408) successfully. The proposed method is very fast, simple, and sensitive without any need for stirring and centrifugation.     

Rapid identification and quantification of methamphetamine and amphetamine in hair by gas chromatography/mass spectrometry coupled with micropulverized extraction,aqueous acetylation and microextraction by packed sorbent

We developed a rapid identification and quantification method for the toxicological analysis of methamphetamine and amphetamine in human hair by gas chromatography/mass spectrometry coupled with a novel combination of micropulverized extraction, aqueous acetylation and microextraction by packed sorbent (MEPS) named MiAMi–GC/MS. A washed hair sample (1–5 mg) was micropulverized for 5 min in a 2 mL plastic tube with 250 μL of water. An anion-exchange sorbent was added to adsorb anionic interferences. After removing the residue with a membrane-filter unit, sodium carbonate and acetic anhydride was admixed in turn. Acetylation was completed in approximately 20 min at room temperature. The acetylated analytes in the reaction liquid were concentrated to an octadecylsilica sorbent packed in the needle of a syringe by a CombiPAL autosampler. Elution was carried out with 50 μL of methanol, and the entire eluate injected into a gas chromatograph using a programmable temperature vaporizing (PTV) technique. The time required for sample preparation and GC/MS analysis was approximately 1 h from a washed hair sample, and an evaporation process was not required. Ranges for quantification were 0.20–50 (ng/mg) each for methamphetamine and amphetamine using 1 mg of hair. Accuracy and relative standard deviation (RSD) were evaluated intraday and interday at three concentrations, and the results were within the limit of a guidance issued by U.S. Food and Drug Administration. For identification, full-scan mass spectra of methamphetamine and amphetamine were obtained using 5 mg of fortified hair samples at 0.2 ng/mg. The extraction device of MEPS was durable for at least 300 extractions, whereas the liner of the gas chromatograph should be replaced after 20–30 times use. The carry over was estimated to be about 1–2%. This sample-preparation method coupled with GC/MS is fast and labor-saving in comparison with conventional methods.     

Miniaturized Laser-Induced Breakdown Spectroscopy for the in-situ analysis of the Martian surface: Calibration and quantification

We report on our ongoing studies to develop Laser-Induced Breakdown Spectroscopy (LIBS) for planetary surface missions to Mars and other planets and moons, like Jupiter's moon Europa or the Earth's moon. Since instruments for space missions are severely mass restricted, we are developing a light-weight miniaturized close-up LIBS instrument to be installed on a lander or rover for the in-situ geochemical analysis of planetary surface rocks and coarse fines. The total mass of the instrument will be ≈ 1 kg in flight configuration. Here we report on a systematic performance study of a LIBS instrument equipped with a prototype laser of 216 g total mass and an energy of 1.8 mJ. The LIBS measurements with the prototype laser and the comparative measurements with a regular 40 mJ laboratory laser were both performed under Martian atmospheric conditions.     

Portable,lightweight, low power,ion chromatographic system with open tubular capillary columns

Basic operation principles of a lightweight, low power, low cost, portable ion chromatograph utilizing open tubular ion chromatography in capillary columns coated with multi-layer polymeric stationary phases are demonstrated. A minimalistic configuration of a portable IC instrument was developed that does not require any chromatographic eluent delivery system, nor sample injection device as it uses gravity-based eluent flow and hydrodynamic sample injection adopted from capillary electrophoresis. As a detection device, an inexpensive commercially available capacitance sensor is used that has been shown to be a suitable substitute for contactless conductivity detection in capillary separation systems. The built-in temperature sensor allows for baseline drift correction typically encountered in conductivity/capacitance measurements without thermostating device. The whole instrument does not require any power supply for its operation, except the detection and data acquisition part that is provided by a USB port of a Netbook computer. It is extremely lightweight, its total weight including the Netbook computer is less than 2.5 kg and it can be continuously operated for more than 8 h. Several parameters of the instrument, such as detection cell design, eluent delivery systems and data treatment were optimized as well as the composition of eluent for non-suppressed ion chromatographic analysis of common inorganic cations (Na⁺, NH₄⁺, K⁺, Cs⁺, Ca²⁺, Mg²⁺, transition metals). Low conductivity eluents based on weakly complexing organic acids such as tartaric, oxalic or pyridine-2,6-dicarboxylic acids were used with contactless capacitance detection for simultaneous separation of mono- and divalent cations. Separation of Na⁺ and NH₄⁺ cations was optimized by addition of 18-crown-6 to the eluent. The best separation of 6 metal cations commonly present in various environmental samples was accomplished in less than 30 min using a 1.75 mM pyridine-2,6-dicarboxylic acid and 3 mM 18-crown-6 eluent with excellent repeatability (below 2%) and detection limits in the low micromolar range. The analysis of field samples is demonstrated; the concentrations of common inorganic cations in river water, mineral water and snow samples were determined.     

Surface-enhanced Raman scattering for perchlorate detection using cystamine-modified gold nanoparticles

Perchlorate (ClO₄⁻) has recently emerged as a widespread environmental contaminant found in groundwater and surface water, and there is a great need for rapid detection and monitoring of this contaminant. This study presents a new technique using cystamine-modified gold nanoparticles as a substrate for surface-enhanced Raman scattering (SERS) detection of perchlorate at low concentrations. A detection limit of 5 × 10⁻⁶ M (0.5 mg/L) has been achieved using this method without sample preconcentration. This result was attributed to a strong plasmon enhancement by gold metal surfaces and the electrostatic attraction of ClO₄⁻ onto positively charged, cystamine-modified gold nanoparticles at a low pH. The methodology also was found to be reproducible, quantitative, and not susceptible to significant interference from the presence of anions such as sulfate, phosphate, nitrate and chloride at concentrations <1 mM, making it potentially suitable for rapid screening and routine analysis of perchlorate in environmental samples.