Integrated microfluidic device with an electroplated palladium decoupler for more sensitive amperometric detection of the 8-hydroxy-deoxyguanosine (8-OH-dG) DNA adduct

8-Hydroxy-deoxyguanosine (8-OH-dG) DNA adduct is one of the most frequently used biomarkers reporting on the oxidative stress that leads to DNA damage. More sensitive and reliable microfluidic devices are needed for the detection of these biomarkers of interest. We have developed a capillary electrophoresis (CE)-based microfluidic device with an electroplated palladium decoupler that provides significantly improved detection limit, separation efficiency, and resolving power. The poly(dimethylsiloxane) (PDMS)/glass hybrid device has fully integrated gold microelectrodes covered in situ with palladium nanoparticles using an electroplating technique. The performance and coverage of the electrodes electroplated with palladium particles were evaluated electrochemically and via scanning electron microscope (SEM) imaging, respectively. The performance of the device was tested and evaluated with different buffer systems, pH values, and electric field strengths. The results showed that this device has significantly improved resolving power, even at separation electric field strengths as high as 600 V cm⁻¹. The detection limit for the 8-OH-dG adduct is about 20 attomoles; the concentration limit is on the order of 100 nM (S/N = 3). A linear response is reported for both 8-OH-dG and dG in the range from 100 nM to 150 μM (≈100 pA μM⁻¹) with separation efficiencies of approximately 120,000–170,000 plates m⁻¹.     

An SPR biosensor for the detection of microcystins in drinking water

A surface plasmon resonance (SPR) biosensor for the detection of microcystins (MCs) in drinking water has been developed. Several assay formats have been evaluated. The selected format is based on a competitive inhibition assay, in which microcystin-LR (MCLR) has been covalently immobilized onto the surface of an SPR chip functionalized with a self-assembled monolayer. The influence of several factors affecting sensor performance, such as the nature and concentration of the antibody, the composition of the carrier buffer, and the blocking and regeneration solutions, has been evaluated. The optimized SPR biosensor provides an IC₅₀ 0.67 ± 0.09 μg L⁻¹, a detection limit of 73 ± 8 ng L⁻¹, and a dynamic range from 0.2 to 2.0 μg L⁻¹ for MCLR. Cross-reactivity to other related MCs, such as microcystin-RR (88%) and microcystin-YR (94%), has also been measured. The SPR biosensor can perform four simultaneous determinations in 60 min, and each SPR chip can be reused for at least 40 assay–regeneration cycles without significant binding capacity loss. The biosensor has been successfully applied to the direct analysis of MCLR in drinking water samples, below the provisional guideline value of 1 μg L⁻¹ established by the World Health Organization for drinking water.     

Optimisation of stir bar sorptive extraction and in-tube derivatisation-thermal desorption-gas chromatography-mass spectrometry for the determination of several endocrine disruptor compounds in environmental water samples

The analysis of organic pollutants in environmental water samples requires a pre-concentration step. Pre-concentration techniques such as stir bar sorptive extraction (SBSE) have gained popularity since they minimise the use of toxic organic solvents and can be considered as green analytical techniques. Similar to other pre-concentration techniques, one of the problems when SBSE is used is the matrix effect, which often occurs during the analysis of environmental water samples such as estuarine or wastewater samples. The present work studied the matrix effect during SBSE coupled to in-tube derivatisation–thermal desorption (TD)–gas chromatography–mass spectrometry for the determination of several endocrine disruptor compounds, such as alkylphenols, bisphenol A, estrogens and sterols, in environmental water samples, after optimisation of the major variables affecting the determination. Variables such as the addition of methanol or an inert salt to the donor phase, the extraction temperature, the volume of the donor phase, the stirring rate and the extraction time were studied during the SBSE optimisation. In the case of the in-tube derivatisation and TD step, the volume of the derivatisation reagent (N,O-bis(trimethylsilyl)triufloroacetamide with 1% of trimethylchlorosilane (BSTFA + 1% TMCS)) and the cryo-focusing temperature were fixed (2 μL and −50 °C, respectively) according to a consensus between maximum signal and optimal operation conditions. Good apparent recovery values (78–124%) were obtained for most of the analytes in Milli-Q water, except for 4-tert-octylphenol (4-tOP), which showed apparent recovery values exceeding 100%. Precision (n = 4) was in the 2–27%, and method detection limits were in the low nanogrammes per litre level for most of the analytes studied. The matrix effect was studied using two different approaches. On the one hand, Milli-Q water samples were spiked with humic acids, and apparent recovery values were studied with and without correction with the corresponding deuterated analogue. On the other hand, estuarine water and wastewater samples were spiked with known concentrations of target analytes, and apparent recoveries were studied as explained above. In general, the matrix effect could be corrected with the use of deuterated analogues, except for 4-tOP and nonylphenols for which [²H₄]-n-nonylphenol did not provide good corrections.     

Integration of spore-based genetically engineered whole-cell sensing systems into portable centrifugal microfluidic platforms

Bacterial whole-cell biosensing systems provide important information about the bioavailable amount of target analytes. They are characterized by high sensitivity and specificity/selectivity along with rapid response times and amenability to miniaturization as well as high-throughput analysis. Accordingly, they have been employed in various environmental and clinical applications. The use of spore-based sensing systems offers the unique advantage of long-term preservation of the sensing cells by taking advantage of the environmental resistance and ruggedness of bacterial spores. In this work, we have incorporated spore-based whole-cell sensing systems into centrifugal compact disk (CD) microfluidic platforms in order to develop a portable sensing system, which should enable the use of these hardy sensors for fast on-field analysis of compounds of interest. For that, we have employed two spore-based sensing systems for the detection of arsenite and zinc, respectively, and evaluated their analytical performance in the miniaturized microfluidic format. Furthermore, we have tested environmental and clinical samples on the CD microfluidic platforms using the spore-based sensors. Germination of spores and quantitative response to the analyte could be obtained in 2.5–3 h, depending on the sensing system, with detection limits of 1 × 10⁻⁷ M for arsenite and 1 × 10⁻⁶ M for zinc in both serum and fresh water samples. Incorporation of spore-based whole-cell biosensing systems on microfluidic platforms enabled the rapid and sensitive detection of the analytes and is expected to facilitate the on-site use of such sensing systems.     

A new bacterial biosensor for trichloroethylene detection based on a three-dimensional carbon nanotubes bioarchitecture

Trichloroethylene (TCE), a suspected human carcinogen, is one of the most common volatile groundwater contaminants. Many different methodologies have already been developed for the determination of TCE and its degradation products in water, but most of them are costly, time-consuming and require well-trained operators. In this work, a fast, sensitive and miniaturised whole cell conductometric biosensor was developed for the determination of trichloroethylene. The biosensor assembly was prepared by immobilising Pseudomonas putida F1 bacteria (PpF1) at the surface of gold interdigitated microelectrodes through a three-dimensional alkanethiol self-assembly monolayer/carbon nanotube architecture functionalised with Pseudomonas antibodies. The biosensor response was linear from 0.07 to 100 μM of TCE (9–13,100 μg L⁻¹). No significant loss of the enzymatic activity was observed after 5 weeks of storage at 4 °C in the M457 pH 7 defined medium (two or three measurements per week). Ninety-two per cent of the initial signal still remained after 7 weeks. The biosensor response to TCE was not significantly affected by cis-1,2-dichloroethylene and vinyl chloride and, in a limited way, by phenol. Toluene was the major interference found. The bacterial biosensor was successfully applied to the determination of TCE in spiked groundwater samples and in six water samples collected in an urban industrial site contaminated with TCE. Gas chromatography–mass spectrometric analysis of these samples confirmed the biosensor measurements.     

Optimization of a miniaturized DBD plasma chip for mercury detection in water samples

In this work, an optimization study was conducted to investigate the performance of a custom-designed miniaturized dielectric barrier discharge (DBD) microplasma chip to be utilized as a radiation source for mercury determination in water samples. The experimental work was implemented by using experimental design, and the results were assessed by applying statistical techniques. The proposed DBD chip was designed and fabricated in a simple way by using a few microscope glass slides aligned together and held by a Perspex chip holder, which proved useful for miniaturization purposes. Argon gas at 75–180 mL/min was used in the experiments as a discharge gas, while AC power in the range 75–175 W at 38 kHz was supplied to the load from a custom-made power source. A UV-visible spectrometer was used, and the spectroscopic parameters were optimized thoroughly and applied in the later analysis. Plasma characteristics were determined theoretically by analysing the recorded spectroscopic data. The estimated electron temperature (T _e = 0.849 eV) was found to be higher than the excitation temperature (T _exc = 0.55 eV) and the rotational temperature (T _rot  = 0.064 eV), which indicates non-thermal plasma is generated in the proposed chip. Mercury cold vapour generation experiments were conducted according to experimental plan by examining four parameters (HCl and SnCl₂ concentrations, argon flow rate, and the applied power) and considering the recorded intensity for the mercury line (253.65 nm) as the objective function. Furthermore, an optimization technique and statistical approaches were applied to investigate the individual and interaction effects of the tested parameters on the system performance. The calculated analytical figures of merit (LOD = 2.8 μg/L and RSD = 3.5%) indicates a reasonable precision system to be adopted as a basis for a miniaturized portable device for mercury detection in water samples.     

Highly sensitive modified glassy carbon sensor based on TDAN for nitrate detection in real water

Glassy carbon electrodes modified with a plasticized PVC membrane including tetradodecyl ammonium nitrate (TDAN) and two different plasticizers [bis(2-etheylhexyl) sebacate (DOS) and dibutyl phthalate (DBP)]. TDAN is a quaternary ammonium salt, used in nitrate-selective field-effect transistor membrane, and it is applied as an ionophore for sensor development. Membranes were prepared as sensing matrix for nitrate detection through the intensity of its reduction peak using cyclic voltammetry. Under optimum conditions, the detection limits of nitrate obtained with TDAN + DOS and TDAN + DBP, are 10–6 mol/dm3 and 10–9 mol/dm3, respectively. As a result, the developed sensors based on TDAN + DOS and TDAN + DBP matrices demonstrate satisfactory quantified amounts in determination of nitrate in water where the measured values were less than 0.57 × 10–7 mol/dm3 and 0.73 × 10–10 mol/dm3, respectively.     

Monitoring of the non-steroid anti-inflammatory drug indomethacin: development of immunochemical methods for its purification and detection

The present research focused on the development of an immunoassay and an immunochemical sol–gel-based immunoaffinity purification (IAP) method for purification and detection of the non-steroid anti-inflammatory drug (NSAID) indomethacin (IMT). A polyclonal antibody (Ab) for IMT was generated, and two sensitive microplate assays for the detection of IMT were developed (termed OV and HRP formats), based on the enzyme-linked immunosorbent assay (ELISA) method. The limits of detection of the assays were 15 ± 1.25 ng mL⁻¹ (n = 50) and 12 ± 0.17 ng mL⁻¹ (n = 4) for the OVA and HRP formats, respectively. The Abs exhibited slight cross-reactivity with other NSAIDs. The Abs were also used to develop a sol–gel-based IAP method for clean-up and concentration of IMT. Several sol–gel formats with various amounts of antibodies were examined; the best and most reproducible format was at a TMOS:HCl molar ratio of 1:6 in which 120 μL of IMT Abs was entrapped. The binding capacity under these conditions was ca. 100 to 250 ng of IMT with very low non-specific binding (less than 5% of the applied amount). The sol–gel IAP method, combined with solid-phase extraction, successfully eliminated serum interference to a degree that enabled analysis of spiked serum samples by ELISA. The method was also found to be fully compatible with subsequent chemical analytical methods, such as liquid chromatography followed by mass spectrometry. The approaches developed in this study form a basis for analysis of IMT in biological samples in order to monitor their pharmacokinetic properties, and may be further used to study population exposure to IMT, and to monitor the occurrence of IMT contamination in water samples.     

Multi-channel PMMA microfluidic biosensor with integrated IDUAs for electrochemical detection

A novel multi-channel poly(methyl methacrylate) (PMMA) microfluidic biosensor with interdigitated ultramicroelectrode arrays (IDUAs) for electrochemical detection was developed. The focus of the development was a simple fabrication procedure and the realization of a reliable large IDUA that can provide detection simultaneously to several microchannels. As proof of concept, five microchannels are positioned over a large single IDUA where the channels are parallel with the length of the electrode finger. The IDUAs were fabricated on the PMMA cover piece and bonded to a PMMA substrate containing the microfluidic channels using UV/ozone-assisted thermal bonding. Conditions of device fabrication were optimized realizing a rugged large IDUA within a bonded PMMA device. Gold adhesion to the PMMA, protective coatings, and pressure during bonding were optimized. Its electrochemical performance was studied using amperometric detection of potassium ferri and ferro hexacyanide. Cumulative signals within the same chip showed very good linearity over a range of 0–38 μM (R ²?=?0.98) and a limit of detection of 3.48 μM. The bonding of the device was optimized so that no cross talk between the channels was observed which otherwise would have resulted in unreliable electrochemical responses. The highly reproducible signals achieved were comparable to those obtained with separate single-channel devices. Subsequently, the multi-channel microfluidic chip was applied to a model bioanalytical detection strategy, i.e., the quantification of specific nucleic acid sequences using a sandwich approach. Here, probe-coated paramagnetic beads and probe-tagged liposomes entrapping ferri/ferro hexacyanide as the redox marker were used to bind to a single-stranded DNA sequence. Flow rates of the non-ionic detergent n-octyl-β-d-glucopyranoside for liposome lysis were optimized, and the detection of the target sequences was carried out coulometrically within 250 s and with a limit of detection of 12.5 μM. The robustness of the design and the reliability of the results obtained in comparison to previously published single-channel designs suggest that the multi-channel device offers an excellent opportunity for bioanalytical applications that require multianalyte detection and high-throughput assays.

Detection of cholera toxin in seafood using a ganglioside-liposome immunoassay

Microbiological contamination of foods continues to be a major concern in public health. Biological toxins are one class of important contaminants that can cause various human diseases. Outbreaks related to contamination by biological toxins or toxin-producing microorganisms have made it extremely important to develop rapid (approximately 20 min), sensitive and cost-effective analytical methods. This paper describes the development of a sensitive bioassay for the detection of cholera toxin (CT) in selected seafood samples, using ganglioside-incorporated liposomes. In this study, the assays were run with food samples spiked with various concentrations of CT. The limit of detection (LOD) increased by a factor of about 10–20 in most food samples, compared with the LOD in the buffer system previously reported. However, the LOD of toxins in food samples (8 × 10–3 × 10³ fg/mL for CT) was still comparable to, or lower than, that previously reported for other assays. The results from this study demonstrate that the bioassays using ganglioside-liposomes can detect the toxin directly in the field screening of food samples rapidly, simply and reliably, without the need for complex instrumentation.     

Integrated Biosensor Systems for Ethanol Analysis

Different integrated systems with a bi-enzymatic biosensor, working with two different methods for ethanol detection—flow injection analysis (FIA) or sequential injection analysis (SIA)—were developed and applied for ethanol extracted from gasohol mixtures, as well as for samples of alcoholic beverages and fermentation medium. A detection range of 0.05–1.5 g ethanol/l, with a correlation coefficient of 0.9909, has been reached when using FIA system, working with only one microreactor packed with immobilized alcohol oxidase and injecting free horseradish peroxidase. When using both enzymes, immobilized separately in two microreactors, the detection ranges obtained varied from 0.001 to 0.066 g ethanol/l, without on-line dilution to 0.010–0.047 g ethanol/l when a 1:7,000 dilution ratio was employed, reaching correlation coefficients of 0.9897 and 0.9992, respectively. For the integrated biosensor SIA system with the stop–flow technique, the linear range was 0.005–0.04 g/l, with a correlation coefficient of 0.9922.     

A microchip sensor for calcium determination

A newly designed glass-PDMS microchip-based sensor for use in the determination of Ca²⁺ ions has been developed, utilizing reflectance measurements from arsenazo III (1,8-dihydroxynaphthalene-3,6-disulfonic acid-2,7-bis[(azo-2)-phenyl arsenic acid]) immobilized on the surface of polymer beads. The beads, produced from cross-linked poly(p-chloromethylstyrene) (PCMS), were covalently modified with polyethylenimine (PEI) to which the Arsenazo III could be adsorbed. The maximum amount of Arsenazo III which could be immobilized onto the PEI-attached PCMS beads was found to be 373.71 mg g⁻¹ polymer at pH 1. Once fabricated, the beads were utilized at the detection point of the microfluidic sensor device with a fiber optic assembly for reflectance measurements. Samples were mobilized past the detection point in the sensor where they interact with the immobilized dye. The sensor could be regenerated and re-used by rinsing with HCl solution. The pH, voltage, linear range, and the effect of interfering ions were evaluated for Ca²⁺ determination using this microchip sensor. At the optimum potential, 0.8 kV, and pH 9.0, the linear range of the microchip sensor was 3.57 × 10⁻⁵ – 5.71 × 10⁻⁴ M Ca²⁺, with a limit of detection (LOD) of 2.68 × 10⁻⁵ M. The microchip biosensor was then applied for clinical analysis of calcium ions in serum with good results. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Development of a novel immunobiosensor method for the rapid detection of okadaic acid contamination in shellfish extracts

The mouse bioassay is the methodology that is most widely used to detect okadaic acid (OA) in shellfish samples. This is one of the best-known toxins, and it belongs to the family of marine biotoxins referred to as the diarrhetic shellfish poisons (DSP). Due to animal welfare concerns, alternative methods of toxin detection are being sought. A rapid and specific biosensor immunoassay method was developed and validated for the detection of OA. An optical sensor instrument based on the surface plasmon resonance (SPR) phenomenon was utilised. A polyclonal antibody to OA was raised against OA–bovine thyroglobulin conjugate and OA–N-hydroxy succinimide ester was immobilised onto an amine sensor chip surface. The assay parameters selected for the analysis of the samples were: antibody dilution, 1/750; ratio of antibody to standard, 1:1; volume of sample injected, 25 μl min⁻¹; flow rate, 25 μl min⁻¹. An assay action limit of 126 ng g⁻¹ was established by analysing of 20 shellfish samples spiked with OA at the critical concentration of 160 ng g⁻¹, which is the action limit established by the European Union (EU). At this concentration of OA, the assay delivered coefficient of variations (CVs) of <10%. The chip surface developed was shown to be highly stable, allowing more than 50 analyses per channel. When the concentrations of OA determined with the biosensor method were compared with the values obtained by LC–MS in contaminated shellfish samples, the correlation between the two analytical methods was found to be highly satisfactory (r ² = 0.991). Figure Biacore     

Development of a microfluidic biosensor module for pathogen detection

The development of a microfluidic biosensor module with fluorescence detection for the identification of pathogenic organisms and viruses is presented in this article. The microfluidic biosensor consists of a network of microchannels fabricated in polydimethylsiloxane (PDMS) substrate. The microchannels are sealed with a glass substrate and packed in a Plexiglas housing to provide connection to the macro-world and ensure leakage-free flow operation. Reversible sealing permits easy disassembly for cleaning and replacing the microfluidic channels. The fluidic flow is generated by an applied positive pressure gradient, and the module can be operated under continuous solution flow of up to 80 microL min(-1). The biosensor recognition principle is based on DNA/RNA hybridization and liposome signal amplification. Superparamagnetic beads are incorporated into the system as a mobile solid support and are an essential part of the analysis scheme. In this study, the design, fabrication and the optimization of concentrations and amounts of the different biosensor components are carried out. The total time required for an assay is only 15 min including sample incubation time. The biosensor module is designed so that it can be easily integrated with a micro total analysis system, which will combine sample preparation and detection steps onto a single chip.     

A lab-on-chip for biothreat detection using single-molecule DNA mapping

Rapid, specific, and sensitive detection of airborne bacteria, viruses, and toxins is critical for biodefense, yet the diverse nature of the threats poses a challenge for integrated surveillance, as each class of pathogens typically requires different detection strategies. Here, we present a laboratory-on-a-chip microfluidic device (LOC-DLA) that integrates two unique assays for the detection of airborne pathogens: direct linear analysis (DLA) with unsurpassed specificity for bacterial threats and Digital DNA for toxins and viruses. The LOC-DLA device also prepares samples for analysis, incorporating upstream functions for concentrating and fractionating DNA. Both DLA and Digital DNA assays are single molecule detection technologies, therefore the assay sensitivities depend on the throughput of individual molecules. The microfluidic device and its accompanying operation protocols have been heavily optimized to maximize throughput and minimize the loss of analyzable DNA. We present here the design and operation of the LOC-DLA device, demonstrate multiplex detection of rare bacterial targets in the presence of 100-fold excess complex bacterial mixture, and demonstrate detection of picogram quantities of botulinum toxoid.     

A microfluidic biosensor based on nucleic acid sequence recognition

The development of a generic semi-disposable microfluidic biosensor for the highly sensitive detection of pathogens via their nucleic acid sequences is presented in this paper. Disposable microchannels with defined areas for capture and detection of target pathogen RNA sequence were created in polydimethylsiloxane (PDMS) and mounted onto a reusable polymethylmethacrylate (PMMA) stand. Two different DNA probes complementary to unique sequences on the target pathogen RNA serve as the biorecognition elements. For signal generation and amplification, one probe is coupled to dye encapsulated liposomes while the second probe is coupled to superparamagnetic beads for target immobilization. The probes hybridize to target RNA and the liposome–target-bead complex is subsequently captured on a magnet. The amount of liposomes captured correlates directly to the concentration of target sequence and is quantified using a fluorescence microscope. Dengue fever virus serotype 3 sequences and probes were used as a model analyte system to test the sensor. Probe binding and target capture conditions were optimized for sensitivity resulting in a detection limit of as little as 10 amol L^–1 (10 pmol L^–1) . Future biosensors will be designed to incorporate a mixer and substitute the fluorescence detection with an electrochemical detection technique to provide a truly portable microbiosensor system.     

Biosensor for on-line fluorescent detection of trifluoroperazine based on genetically modified calmodulin

This paper describes the development of a novel on-line biosensor based on a fluorescently labeled human calmodulin (CaM), hCaM M124C-mBBr, immobilized on controlled-pore glass (CPG), for the analysis of trifluoroperazine (TFP); a phenothiazine drug in human urine samples. The device was automated by packing hCaM M124C-mBBr-CPG in a continuous-flow microcell connected to a monitoring system, composed of a bifurcated optical fiber coupled to a spectrofluorometer. Operating parameters of the on-line biosensor (flow rate, sample injection volume, and carrier solution and buffer pH) were studied and optimized. Under the optimal conditions, the biosensor provides a detection and a quantification limit of 0.24 and 0.52 μg mL⁻¹, respectively, and a dynamic range from 0.52 to 61.05 μg mL⁻¹ TFP (n = 5, correlation coefficient 0.998). The response time (t ₁₀₀) was shorter than 42 s (recovery time <4.5 min) and reproducibility and repeatability of the TFP measurements, within the linear response range, were lower than 1.4 and 2.7%, respectively. The device was successfully applied to the analysis of TFP in spiked human urine samples with recoveries ranging between 97 and 101% and with RSDs lower than 5.9%.     

Capacitive biosensor for detection of endotoxin

A capacitive biosensor for the detection of bacterial endotoxin has been developed. Endotoxin-neutralizing protein derived from American horseshoe crab was immobilized to a self-assembled thiol layer on a biosensor transducer (Au). Upon injection of a sample containing endotoxin, a decrease in the observed capacitive signal was registered. Endotoxin could be determined under optimum conditions with a detection limit of 1.0 × 10⁻¹³ M and linearity ranging from 1.0 × 10⁻¹³ to 1.0 × 10⁻¹⁰ M. Good agreement was achieved when applying endotoxin preparations purified from an Escherichia coli cultivation to the capacitive biosensor system, utilizing the conventional method for quantitative endotoxin determination, the Limulus amebocyte lysate test as a reference. The capacitive biosensor method was statistically tested with the Wilcoxon signed rank test, which proved the system is acceptable for the quantitative analysis of bacterial endotoxin (P < 0.05). Figure The flow-injection capacitive biosensor system and the capacitive properties of the transducer surface, where C_SAM is the capacitance change of the self-assembled thiol monolayer, C_P is the capacitance change of the protein layer, C_a is the capacitance change of the analyte layer and C_Total is the total capacitance change measured at the working electrode/solution interface (modified from Limbut et al., 2006. Biosens Bioelectron 22: 233-240)     

Determination of cocaine on banknotes through an aptamer-based electrochemiluminescence biosensor

A novel electrochemiluminescence (ECL) “sandwich” biosensor has been developed to detect cocaine. The sandwich biosensor was fabricated on the basis of the fact that a single aptamer could be split into two fragments and the two dissociated parts could form a folded, associated complex in the presence of targets. One of these (capture probe), which had hexane–thiol at its 5′-terminus, was immobilized on a gold electrode via thiol–gold binding. The other one (detection probe) was labeled with the ECL reagent tris(2,2′-bipyridyl)ruthenium(II)-doped silica nanoparticles (RuSiNPs) at its 3′-terminus. Owing to the weak interaction between the two fragments, the sensor exhibited a low ECL signal in the absence of cocaine. After the target cocaine had been added to the solution, it induced association of the two fragments and stabilized the associated complexes, leading to immobilization of RuSiNPs on the electrode surface, and the ECL detected on the electrode surface was enhanced. The enhanced ECL intensity was directly proportional to the logarithm of the cocaine concentration in the range from 1.0 × 10⁻⁹ to1.0 × 10⁻¹¹ mol/L, with a detection limit of 3.7 × 10⁻¹² mol/L. The biosensor was applied to detect trace amounts of cocaine on banknotes with satisfactory results.     

A microfluidic device with integrated fluorimetric detection for flow injection analysis

This work describes the development of flow analysis microsystems with integrated fluorimetric detection cells. Channels (width of 300–540 μm and depth of 200–590 μm) were manufactured by deep-UV lithography in urethane–acrylate (UA) resin. Plastic optical fibers (diameter of 250 μm) were coupled to a 2.0-mm-long detection channel in order to guide the excitation radiation from an LED (470 nm) and collect the emitted radiation at a right angle towards a photomultiplier. A single-line miniaturized system, with a total internal volume of 10.4 μL, was evaluated by means of standard fluorescein solutions (0.53–2.66 μmol L⁻¹, pH 8.5). The analytical signals presented a linear relationship in the concentration range studied, with a relative standard deviation of 1.9% (n = 5), providing a detection limit of 0.37 μmol L⁻¹ and an analytical frequency of 60 samples/h, using a flow rate of 60 μL min⁻¹. Optical microscopy images and videos acquired in real time for the hydrodynamic injection of 130 and 320 nL of sample solutions indicated the good performance of the proposed sampling strategy. Another microsystem with a total internal volume of 38 μL was developed, incorporating a confluence point for two solutions. This device was applied to the determination of the total concentration of Ca²⁺ and Mg²⁺ in commercial mineral waters using the calcein method. Microscopy images and videos demonstrated the mixing efficiency of the solutions in the microchannels. A linear relationship was observed for the analytical signal in the Ca²⁺ concentration range from 25 to 125 μmol L⁻¹, with relative standard deviations of 3.5%. The analysis of mineral waters with the proposed system provided results that did not differ significantly from those obtained by the EDTA titration method at a confidence level of 95%. These results demonstrate the viability of developing micro flow injection systems with an integrated fluorimetric detection cell.