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.     

Flow injection on-line dilution for flame atomic absorption spectrometry by micro-sample introduction and dispersion using syringe pumps

A robust flow injection (FI) on-line dilution system based on micro-sample introduction was developed for flame atomic absorption spectrometry (FAAS). Two computer programmed and stepper-motor driven syringe pumps were used for the precise and reproducible sample metering in micro-liters and carrier delivery. Factors, which might influence the performance of the system, such as sample matrix and carryover, were investigated. No inferior effects were observed with various matrices including 10% glycerol. Sample carryover effects were less than 0.4%, tested by analyzing a blank and a sample alternately. Dilution factors were decided and keyed in manually. The system was calibrated using a set of concentrated standard solutions for a given dilution factor. At a sampling frequency of 60 h⁻¹, precisions were better than 2% R.S.D. (n=40) for dilution factors of 10-2000. The long-term stability of the system was examined by continuously running the system for a whole working day, and a precision of 2.6% R.S.D. (n=345) was obtained at a dilution factor of 1000. The system was verified by analyzing a standard copper alloy with a certified concentration of 57.4% Cu, resulting in a measurement solution with 574 mg l⁻¹ Cu.     

Development of an on-line isotope dilution laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) method for determination of boron in silicon wafers

A method has been developed based on an on-line isotope dilution technique couple with laser ablation/inductively coupled plasma mass spectrometry (LA-ICP-MS), for the determination of boron in p-type silicon wafers. The laser-ablated sample aerosol was mixed on-line with an enriched boron aerosol supplied continuously using a conventional nebulization system. Upon mixing the two aerosol streams, the isotope ratio of boron changed rapidly and was then recorded by the ICP-MS system for subsequent quantification based on the isotope dilution principle. As an on-line solid analysis method, this system accurately quantifies boron concentrations in silicon wafers without the need for an internal or external solid reference standard material. Using this on-line isotope dilution technique, the limit of detection for boron in silicon wafers is 2.8 × 10¹⁵ atoms cm⁻³. The analytical results obtained using this on-line methodology agree well with those obtained using wet chemical digestion methods for the analysis of p-type silicon wafers containing boron concentrations ranging from 1.0 × 10¹⁶ to 9.6 × 10¹⁸ atoms cm⁻³.     

Biosensing in microfluidic channels using fluorescence polarization

Microfabricated microfluidic devices provide useful platforms for sensing and conducting immunoassays for high throughput screening and drug discovery. In this paper, fluorescence polarization (FP) has been used as a technique for probing binding events within 500 μm and smaller microfluidic channels fabricated in polydimethylsiloxane. The binding of concanavalin A to a lectin-dextran and a glycoprotein-acetylcholinesterase has been used to demonstrate the homogeneous, ratioing format of fluorescence polarization for the quick and accurate determination of extremely low concentrations. Concentrations of concanavalin A in the 0.2-1.0 nmole range were detected within 500 μm channels. Polarization has also been used to sense for a polyaromatic hydrocarbon (PAH) within a microfluidic channel using binding to a TRITC-labeled antibody. Specifically, concentrations of pyrene in a 10-40 nmole range were sensed in 500 μm microfluidic channels. We have also demonstrated a simple pH sensor based on the change in anisotropy of a pH sensitive fluorophore-SNAFL. The ease of fabrication and measurement using such polarization-based devices make them extremely suitable for micro-sized sensors, assays and total analysis systems.     

Fungal pathogenic nucleic acid detection achieved with a microfluidic microarray device

Detection of polymerase chain reaction (PCR) products obtained from cultured greenhouse fungal pathogens, Botrytis cinerea and Didymella bryoniae has been achieved using a previously developed microfluidic microarray assembly (MMA) device. The flexible probe construction and rapid DNA detection resulted from the use of centrifugal pumping in the steps of probe introduction and sample delivery, respectively. The line arrays of the oligonucleotide probes were “printed” on a CD-like glass chip using a polydimethylsiloxane (PDMS) polymer plate with radial microfluidic channels, and the sample hybridizations were conducted within the spiral channels on the second plate. The experimental conditions of probe immobilization and sample hybridization were optimized, and both complementary oligonucleotides and PCR products were tested. We were able to achieve adequate fluorescent signals with a sample load as small as 0.5 nM (1 μL) for oligonucleotide samples; for PCR products, we achieved detection at the level of 3 ng.     

Fabrication of dissolved O2 metric uric acid biosensor using uricase epoxy resin biocomposite membrane

Uricase purified from 20-day-old leaves of cowpea was immobilized on to epoxy resin membrane with 80% retention of initial activity of free enzyme and a conjugation yield of 0.056 mg/cm². The uricase epoxy resin bioconjugate membrane was mounted over the sensing part of the combined electrode of ‘Aqualytic’ dissolved O₂ (DO) meter to construct a uric acid biosensor. The biosensor measures the depletion of dissolved O₂ during the oxidation of uric acid by immobilized uricase, which is directly proportional to uric acid concentration. The biosensor showed optimum response within 10-12 s at a pH 8.5 and 35 °C. A linear relationship was found between uric acid concentration from 0.025 to 0.1 mM and O₂ (mg/l) consumed. The biosensor was employed for measurement of uric acid in serum. The mean value of uric acid in serum was 4.92 mg/dl in apparently healthy males and 3.11 mg/dl in apparently healthy females. The mean analytic recoveries of added uric acid in reaction mixture (8.9 and 9.8 mg/dl) were 93.6 ± 2.34 and 87.18 ± 3.17% respectively. The within and between batch CVs were <6.5 and <5.0%, respectively. The serum uric acid values obtained by present method and standard enzymic colorimetric method, showed a good correlation (r = 0.996) and regression equation being y = 0.984x + 0.0674. Among the various metabolites tested only, glucose (11%), urea (38%), NaCl (25%) and cholesterol (13%) and ascorbic acid (56%) caused decrease, while, MgSO₄ and CaCl₂ had no effect on immobilized enzyme. The enzyme electrode showed only 32% decrease during its use for 100 times over a period of 60 days at 4 °C.     

Improvement in the sensitivity of microfluidic ELISA through field amplified stacking of the enzyme reaction product

In this article, we demonstrate a novel approach to enhancing the sensitivity of enzyme-linked immunosorbent assays (ELISA) through pre-concentration of the enzyme reaction product (resorufin/4-methylumbelliferone) in free solution. The reported pre-concentration was accomplished by transporting the resorufin/4-methylumbelliferone molecules produced in the ELISA process towards a high ionic-strength buffer stream in a microfluidic channel while applying a voltage drop across this merging region. A sharp change in the electric field around the junction of the two liquid streams was observed to abruptly slow down the negatively charged resorufin/4-methylumbelliferone species leading to the reported pre-concentration effect based on the field amplified stacking (FAS) technique. It has been shown that the resulting enhancement in the detectability of the enzyme reaction product significantly improves the signal-to-noise ratio in the system thereby reducing the smallest detectable analyte concentration in the ELISA method. Applying the above-described approach, we were able to detect mouse anti-BSA and human TNF-α at concentrations nearly 60-fold smaller than that possible on commercial microwell plates. For the human TNF-α sample, this improvement in assay sensitivity corresponded to a limit of detection (LOD) of 0.102 pg mL⁻¹ using the FAS based microfluidic ELISA method as compared to 7.03 pg mL⁻¹ obtained with the traditional microwell plate based approach. Moreover, because our ELISAs were performed in micrometer sized channels, they required sample volumes about two orders of magnitude smaller than that consumed in the latter case (1 μL versus 100 μL).     

Immuno-capture and in situ detection of Salmonella typhimurium on a novel microfluidic chip

The new method presented in this article achieved the goal of capturing Salmonella typhimurium via immunoreaction and rapid in situ detection of the CdSe/ZnS quantum dots (QDs) labeled S. typhimurium by self-assembly light-emitting diode-induced fluorescence detection (LIF) microsystem on a specially designed multichannel microfluidic chip. CdSe/ZnS QDs were used as fluorescent markers improving detection sensitivity. The microfluidic chip developed in this study was composed of 12 sample channels, 3 mixing zones, and 6 immune reaction zones, which also acted as fluorescence detection zones. QDs–IgG–primary antibody complexes were generated by mixing CdSe/ZnS QDs conjugated secondary antibody (QDs–IgG) and S. typhimurium antibody (primary antibody) in mixing zones. Then, the complexes went into immune reaction zones to label previously captured S. typhimurium in the sandwich mode. The capture rate of S. typhimurium in each detection zone was up to 70%. The enriched QDs-labeled S. typhimurium was detected using a self-assembly LIF microsystem. A good linear relationship was obtained in the range from 3.7 × 10 to 3.7 × 10⁵ cfu mL⁻¹ using the equation I = 0.1739 log (C) − 0.1889 with R² = 0.9907, and the detection limit was down to 37 cfu mL⁻¹. The proposed method of online immunolabeling with QDs for in situ fluorescence detection on the designed multichannel microfluidic chip had been successfully used to detect S. typhimurium in pork sample, and it has shown potential advantages in practice.     

Application of octadecanethiol self-assembled monolayer to cholesterol biosensor based on surface plasmon resonance technique

Octadecanethiol (ODT) self-assembled monolayer (SAM) prepared onto gold-coated glass plate has been modified by using nitrene reaction of 1-fluoro-2-nitro-4-azidobenzene (FNAB) that further covalently binds to cholesterol oxidase (ChOx) via thermal reaction. FNAB acts as a bridge (cross-linker) between SAM and ChOx. The ChOx/FNAB/ODT/Au electrode thus fabricated has been characterized using contact angle (CA) measurements, UV-vis spectroscopy, electrochemical techniques and X-ray photoelectron spectroscopy (XPS) technique, respectively. This ChOx/FNAB/ODT/Au bioelectrode has been utilized for estimation of cholesterol in solution using surface plasmon resonance (SPR) technique. This SPR based cholesterol biosensor has linearity from 50 to 500 mg/dl of cholesterol in solution with lower detection limit of 50 mg/dl and shelf life of about 2 months when stored at 4 °C.     

Exploiting the bead injection LOV approach to carry out spectrophotometric assays in wine: application to the determination of iron

A sequential injection lab-on-valve (SI-LOV) system was used to develop a new methodology for the determination of iron in wine samples exploiting the bead injection (BI) concept for solid phase extraction and spectrophotometric measurement. Nitrilotriacetic Acid (NTA) Superflow resin was used to build the bead column of the flow through sensor. The iron (III) ions were retained by the bead column and react with SCN⁻ producing an intense red colour. The change in absorbance was monitored spectrophotometrically on the optosensor at 480 nm. It was possible to achieve a linear range of 0.09-5.0 mg L⁻¹ of iron, with low sample and reagent consumption; 500 μL of sample, 15 μmol of SCN⁻, and 9 μmol of H₂O₂, per assay. The proposed method was successfully applied to the determination of iron in wine, with no previous treatment other than dilution, and to other food samples.     

Direct quantification of creatinine in human urine by using isotope dilution extractive electrospray ionization tandem mass spectrometry

Urinary creatinine (CRE) is an important biomarker of renal function. Fast and accurate quantification of CRE in human urine is required by clinical research. By using isotope dilution extractive electrospray ionization tandem mass spectrometry (EESI–MS/MS) a high throughput method for direct and accurate quantification of urinary CRE was developed in this study. Under optimized conditions, the method detection limit was lower than 50 μg L⁻¹. Over the concentration range investigated (0.05–10 mg L⁻¹), the calibration curve was obtained with satisfactory linearity (R² = 0.9861), and the relative standard deviation (RSD) values for CRE and isotope-labeled CRE (CRE-d3) were 7.1–11.8% (n = 6) and 4.1–11.3% (n = 6), respectively. The isotope dilution EESI–MS/MS method was validated by analyzing six human urine samples, and the results were comparable with the conventional spectrophotometric method (based on the Jaffe reaction). Recoveries for individual urine samples were 85–111% and less than 0.3 min was taken for each measurement, indicating that the present isotope dilution EESI–MS/MS method is a promising strategy for the fast and accurate quantification of urinary CRE in clinical laboratories.     

An integrated digital microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids

Clinical diagnostics is one of the most promising applications for microfluidic lab-on-a-chip systems, especially in a point-of-care setting. Conventional microfluidic devices are usually based on continuous-flow in microchannels, and offer little flexibility in terms of reconfigurability and scalability. Handling of real physiological samples has also been a major challenge in these devices. We present an alternative paradigm--a fully integrated and reconfigurable droplet-based "digital" microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids. The microdroplets, which act as solution-phase reaction chambers, are manipulated using the electrowetting effect. Reliable and repeatable high-speed transport of microdroplets of human whole blood, serum, plasma, urine, saliva, sweat and tear, is demonstrated to establish the basic compatibility of these physiological fluids with the electrowetting platform. We further performed a colorimetric enzymatic glucose assay on serum, plasma, urine, and saliva, to show the feasibility of performing bioassays on real samples in our system. The concentrations obtained compare well with those obtained using a reference method, except for urine, where there is a significant difference due to interference by uric acid. A lab-on-a-chip architecture, integrating previously developed digital microfluidic components, is proposed for integrated and automated analysis of multiple analytes on a monolithic device. The lab-on-a-chip integrates sample injection, on-chip reservoirs, droplet formation structures, fluidic pathways, mixing areas and optical detection sites, on the same substrate. The pipelined operation of two glucose assays is shown on a prototype digital microfluidic lab-on-chip, as a proof-of-concept.     

An environmental friendly method for the automatic determination of hypochlorite in commercial products using multisyringe flow injection analysis

A multisyringe flow injection analysis system was used for the determination of hypochlorite in cleaning agents, by measurement of the native absorbance of hypochlorite at 292 nm. The methodology was based on the selective decomposition of hypochlorite by a cobalt oxide catalyst giving chloride and oxygen. The difference of the absorbance of the sample before and after its pass through a cobalt oxide column was selected as analytical signal. As no further reagent was required this work can be considered as a contribution to environmental friendly analytical chemistry. The entire analytical procedure, including in-line sample dilution in three steps was automated by first, dilution in a stirred miniature vessel, second by dispersion and third by in-line addition of water using multisyringe flow injection technique. The dynamic concentration range was 0.04-0.78 g L⁻¹ (relative standard deviation lower than 3%), where the extension of the hypochlorite decomposition was of 90 ± 4%. The proposed method was successfully applied to the analysis of commercial cleaning products. The accuracy of the method was established by iodometric titration.     

Exploiting the bead injection concept for sequential determination of copper and mercury ions in river-water samples

A procedure involving bead-injection concept and sequential determination of copper and mercury ions in river-water samples is proposed. The method is based on the solid-phase extraction of both metal ions on the same beads surface (Chelex 100 resin) and in their subsequent reaction with the colorimetric reagents (APDC and Dithizone for copper and mercury ions, respectively). For this task, a resin mini-column is established in the optical path by the selection, introduction and trapping of a defined volume of the Chelex-100 resin beads suspension in the flow system. The passage of the sample solution through the resin mini-column promotes the sorption of Cu(II) ions and, making the APDC colorimetric reagent flows through the beads, the formation of the coloured complex on the solid phase surface occurs. The absorbance of the formed APDC-Cu complex is then monitored at 436 nm and the spent beads are discarded. Packing another resin mini-column in the flow cell and repeating the concentration step it is possible to carried out the mercury determination by using Dithizone as reagent. The absorbance of the Dithizone-Hg complex is monitored at 500 nm. After each measurement, the spent beads are wasted and a new portion of fresh one is trapped in the system, letting it ready for the next measurement. The bead injection system is versatile and can be used to concentrate different sample volumes, which permits the determination of a wide range of copper and mercury ions concentrations. When the sample-selected volumes are 100 and 1000 μl the analytical ranges were 5.0 up to 500.0 μg l⁻¹ and 2.5 up to 30.0 μg l⁻¹ for Cu(II) and Hg(II) ions, respectively. Under these conditions, the detection limit was estimated as 0.63 and 0.25 μg l⁻¹ for copper and mercury ions determination. The system consumes 2 mg of Chelex 100 resin beads, 0.20 mg of APDC or 1.25 mg of Dithizone per determination and the traditional organic solvent extraction methodology, normally used in connection with APDC and Dithizone reagents, is not used here which permits to classify the present method as green.     

A new functionalized resin and its application in preconcentration system with multivariate optimization for nickel determination in food samples

In this work, Amberlite XAD-2 resin functionalized with 4,5-dihydroxy-1,3-benzenedisulfonic acid was synthesized, characterized and applied as a new packing material for an on-line system to nickel preconcentration. The method is based on the sorption of Ni(II) ions in a minicolumn containing the synthesized resin, posterior desorption using an acid solution and measurement of the nickel by spectrophotometry (PAR method). The optimization of the system was performed using factorial design and Doehlert matrix considering five variables: eluent concentration, PAR solution pH, sample flow rate, PAR solution concentration and sample pH. Signals were measured as peak height by using an instrument software. Using the experimental conditions defined in the optimization, the method allowed nickel determination with achieved sampling rate of 25 samples per hour, detection limit (3 s) of 2 μg l⁻¹ and precision (assessed as the relative standard deviation) of 8.2-2.6%, for nickel solutions of 10.0-200.0 μg l⁻¹ concentration, respectively. The experimental enrichment factor of the proposed system was 46, for 120 s preconcentration time. The proposed procedure was applied for nickel determination in food samples. Recoveries of spike additions (5 or 10 μg g⁻¹) to food samples were quantitative (94-110%).     

Determination of hypochlorite in water using a chemiluminescent test strip

We have developed a selective and reusable chemiluminescent test strip to determine hypochlorite. The hypochlorite-sensitive test strip contains a 10 mm × 9 mm piece of anionic cellulose paper in fluoresceinate cycle, glued to a 10 mm × 4 cm × 0.5 mm polyester strip. The measurement of the chemiluminescence in a luminometer when 1 ml of sample is injected into a conventional cell containing the strip makes it possible to determine hypochlorite. The composition of the membrane and reaction conditions have been adjusted to obtain adequate sensitivity and selectivity. The test strip responded linearly to hypochlorite over two linear ranges, the first 2.0-10.3 mg L⁻¹ and the second 10.3-51.4 mg L⁻¹, with a detection limit of 0.4 mg L⁻¹. The reproducibility using the same disposable test strip at a medium level of the range was 6.6%, as relative standard deviation (R.S.D.), and 12.3% using different test strips. The procedure was applied to the determination of hypochlorite in different types of waters.     

Exploiting monosegmented flow analysis to perform in-line standard additions using a single stock standard solution in spectrophotometric sequential injection procedures

The robustness of sequential injection analysis (SIA) was combined with the monosegmented flow analysis (MSFA) approach, in which there is no dispersion of the reaction zone with carrier, to develop a methodology to perform in-line dilution. This approach allows one to know accurately the dilution of sample and reagent inside the monosegment, without the need for determination of dispersion coefficients. As a consequence, the methodology allowed the mechanization of procedures to perform standard additions and to construct analytical curves using a single stock standard solution, with very simple and conventional computation of the sample concentration. The method was illustrated with experiments using the bromothymol blue (BTB) dye, in which no reactions are involved, as well as with the spectrophotometric methodology for determination of Fe(II) using o-1,10-phenanthroline as chromogenic reagent. The resulting method presented a sampling frequency of 30 analyses per hour and a detection limit of 25 μg l⁻¹.     

Sequential injection analyzer for glycerol monitoring in yeast cultivation medium

A smart and versatile flow system for the at-line monitoring of glycerol based on sequential injection analysis is proposed. Formaldehyde, generated by oxidation of glycerol with sodium periodate, is transformed into 2,4-diacetyl-1,4-dihydrolutidine applying the Hantzsch condensation reaction with acetylacetone and ammonium. Dual-wavelength detection was carried out to minimize the contribution of the schlieren effect using a single blue LED. In-line sample dilution is accomplished applying the concept of zone-penetration and a new concept of sample splitting. Under optimized physical and chemical variables, regression curves over two dynamic working ranges of 0.1-4 and 1-40 g l⁻¹ were attained. The injection throughputs were 14 and 12 h⁻¹, respectively. Applying on-line data evaluation and conditional inquiries, the smart and independent selection of the adequate analytical procedure for the required working range was accomplished. The system was successfully applied to the at-line monitoring of glycerol in a continuous, cell-free medium flow from a yeast cultivation process during batch and fed-batch phase with glycerol as the only carbon source.     

Enzymatic chemiluminescent assay of glucose by sequential-injection analysis with soluble enzyme and on-line sample dilution

This work reports a sequential-injection analysis (SIA) method for the enzymatic assay of glucose with soluble glucose oxidase (GOD) and on-line sample dilution with chemiluminescence (CL) detection. A zone of sample was aspirated in the holding coil of the SIA manifold and, if necessary, was diluted on-line by means of an auxiliary dilution conduit. Then, a zone of GOD was aspirated adjacent to the sample zone and a stopped-flow period was applied to allow the enzymatic reaction to proceed with production of hydrogen peroxide. Then, zones of a catalyst (Co(II) solution) and alkaline luminol were aspirated into the holding coil. Finally, the flow was reversed and the stacked zones were sent to a flow-cell located in front of a photomultiplier tube (PMT) that monitored the CL intensity. The linear dynamic range was 1 × 10⁻⁵-1 × 10⁻³ mol L⁻¹ glucose, the coefficient of variation at 8 × 10⁻⁵ mol L⁻¹ of glucose was s_r = 3.1% (n = 8), the limit of detection at the 3σ level was c_L = 1 × 10⁻⁶ mol L⁻¹ and the sampling frequency was 28 h⁻¹. With on-line dilution by a factor of 1/200, the linear range could be extended up to 0.2 mol L⁻¹ glucose. The advantages of the proposed method are the simple manifold and instrumentation used, the scope for automated on-line dilution, the low consumption of sample and reagents and the elimination of enzyme immobilisation procedures. The method was applied to the analysis of commercial drinks and honey with percent relative errors in glucose determination in the range 100 ± 6.1%.     

Preliminary study of a microbeads based histamine detection for food analysis using thermostable recombinant histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007

We designed and prepared a micro biosensing system consisting of a flow through system with a sub-micro liter injection valve and a sub-micro liter volume bioreactor. An electrochemical detector was combined with the reactor for immediate detections. The volumes of the reactor and the sample loop for the injection were 850 nl and 320 nl, respectively. This paper described about the characteristics of the sensing system in the case of histamine detection for food analysis. Histamine oxidase from KAIT-B-007 was prepared by using a gene recombination technique and they were immobilized with chitosan beads (? = 70-105 μm). The detection less than one minute after injection made possible fast analysis for histamine. The biosensing system also showed a high performance for histamine detection in wide range of 1 μM-1 mM. In addition, we practically measured histamine content in raw tuna stored at room temperature and 35 °C up to 96 h. As a result of the comparison between our sensing system and HPLC method, there was good agreement. These results show that our microfluidic biosensing system has the potential to assist miniaturization with small sample volume and short determination time for a sequential food analysis.