Reflectometric interference spectroscopy (RIfS) as a new tool to measure in the complex matrix milk at low analyte concentration

Measurements in complex matrices like milk still present a challenge in biosensor development. This is especially important when using a label-free detection method or when measuring low analyte concentrations. The direct optical method reflectometric interference spectroscopy (RIfS) was used for investigating matrix effects in immunoassay development. Furthermore, approaches to reduce these effects have been established. As a model system, the hormone testosterone has been chosen because this immunoassay has been well characterized in buffer. In a first step, the immunoassay for the detection of testosterone in buffer was improved beyond former published results. Therefore, the sensor surface was optimized, resulting in a fivefold lower limit of detection (70.2 ng L⁻¹) and limit of quantification (130.0 ng L⁻¹). Additionally, the assay time could be reduced to 15 min. Consequently, we used this improved assay to investigate matrix effects of whole pasteurized bovine milk. To minimize these effects, the surface chemistry was adapted and a suitable evaluation method was established, reducing the effects of Tyndall scattering and nonspecific binding to the sensor surface. These improvements allow for very reliable quantitative measurements in milk. The assay developed required no sample pretreatment and allowed for the regeneration of the sensor surface so that calibration could be performed on one chip. The calibration in milk (3.5% fat) resulted in a limit of detection of 94.4 ng L⁻¹ and a limit of quantification of 229.3 ng L⁻¹. Furthermore, recovery rates between 70% and 120% could be obtained. Thus, for the first time, an analyte in the matrix milk was successfully quantified with RIfS at low concentrations.     

Prostate-specific antigen immunoassays on the BioCD

The BioCD is a spinning-disc interferometric biosensor on which antibodies are immobilized to capture target antigens from biological samples. In this work, BioCDs measured the interferometric response to prostate-specific antigen (PSA). The ideal detection limit for PSA was determined using a BioCD with 12,500 printed target antibody spots with a corresponding number of reference protein spots. Statistical analysis projects the detection limit of PSA as a function of the number of spots included in the average. When approximately 10,000 spot pairs were averaged, the 3σ detection limit was 60 pg/ml in a 2 mg/ml simple protein background. A standard format for BioCD immunoassays uses 96 wells with 32 target spots paired with reference spots. In serum, the detection limit for this format was 1 ng/ml in 3:1 diluted female human serum using a sandwich assay with a nonfluorescent mass tag.     

Polymer waveguide backplanes for optical sensor interfaces in microfluidics

A polymer optical backplane capable of generic luminescence detection within microfluidic chips is demonstrated using large core polymer waveguides and vertical couplers. The waveguides are fabricated through a new process combining mechanical machining and vapor polishing with elastomer microtransfer molding. A backplane approach enables general optical integration with planar array microfluidics since optical backplanes can be independently designed but still integrated with planar fluidic circuits. Fabricated large core waveguides exhibit a loss of 0.1 dB cm(-1) at 626 nm, a measured numerical aperture of 0.50, and a collection efficiency of 2.86% in an n = 1.459 medium, comparable to a 0.50 NA microscope objective. In addition to vertical couplers for out-of-plane collection and excitation, polymer waveguides are doped with organic dyes to provide wavelength selective filtering within waveguides, further improving optical device integration. With large core low loss waveguides, luminescence collection is improved and measurements can be performed with simple LEDs and photodetectors. Fluorescein detection via fluorescence intensity with a limit of detection (3sigma) of 200 nM in a 1 microL volume is demonstrated. Phosphorescence lifetime based oxygen detection in water in an oxygen controllable microbial cell culture chip with a limit of detection (3sigma) of 0.08% or 35 ppb is also demonstrated utilizing the waveguide backplane. Single waveguide luminescence collection performance is equivalent to a back collection geometry fiber bundle consisting of nine 500 microm diameter collection fibers.     

Development of an enzyme-linked immunosorbent assay for metolcarb residue analysis and investigation of matrix effects from different agricultural products

The development of a direct competitive enzyme-linked immunosorbent assay based on polyclonal antibodies for N-methylcarbamate insecticide metolcarb is described. Two new haptens for the metolcarb were designed and synthesized. Both haptens were conjugated with keyhole limpet hemocyanin to form the immunogens. Four rabbits were immunized with the immunogens for production of polyclonal antibodies against metolcarb. Antisera titers were tested on the homologous coating antigens using a noncompetitive indirect enzyme-linked immunosorbent assay. The high titer antisera were used to develop the direct competitive enzyme-linked immunosorbent assay for the detection of metolcarb. The antibody–antigen combination with the highest selectivity for metolcarb was further optimized and its tolerance to changes in chemical conditions (ionic strength, pH value, and organic solvent) was studied. Under optimum conditions, the sensitivity and the limit of detection were determined to be 22 μg L⁻¹ and 1.2 μg L⁻¹ respectively. Determination of metolcarb in fruit juices and vegetables was accomplished by simple, rapid, and efficient extraction methods. Recoveries of metolcarb from spiked samples ranged from 80.5% to 109.5%. Validation of the developed immunosorbent assay was conducted by comparison of results from high-performance liquid chromatography. The correlation between the data obtained using developed immunosorbent assay and high-performance liquid chromatography was high (R ² = 0.9884). Therefore, the developed immunosorbent assay in this study was suitable for the rapid quantitative determination of metolcarb in agricultural products.     

Ultrasensitive polyaniline-nickel oxide cladding modified with urease immobilized intrinsic optical fiber urea biosensor

Herein, we report a polyaniline-nickel oxide (PANI-NiO) nanocomposite as an efficient immobilization matrix for development the optical fiber urea biosensor. Optical fiber sensing probe was developed by removing some portion of optical fiber at middle and modified with PANI-NiO matrix. After the modification of cladding removed portion, it was immobilized with enzyme urease via glutaraldehyde as a bi-functional cross-linking agent. The physicochemical and optical properties of the PANI-NiO matrix were explored by X-ray diffraction, scanning electron microscopy, ultraviolet–visible, and Fourier transform infrared spectroscopic techniques. The characteristic features and performance of the developed sensor were evaluated via recording the output power and modal power distribution by means of a charge-coupled device camera. The developed urea biosensor exhibits a selective response towards urea concentrations in the linear range 1 nM–100 mM with a lower detection limit of 1 nM. Sensor recorded as a 40 days stability and response time ~1 min. Thus, the obtained experimental results of the developed sensor promote its applicability with practical prospects in diverse field.     

A new procalcitonin optical immunosensor for POCT applications

A new immunosensor for the determination of procalcitonin was developed. A sandwich assay format was implemented on a polymethylmetacrylate optical biochip, opportunely shaped in order to obtain several flow channels and potentially suitable for point of care testing applications. The sandwich format makes use of two new rat monoclonal antibodies. The capture antibody was covalently immobilised on the surface of the plastic chip, and the detection antibody was labelled with DY647 dye. Different combinations of capture and detection antibodies were investigated, and particular attention was devoted in order to avoid the non-specific adsorption. A limit of detection of 0.088 mg L⁻¹ was achieved within the working range of 0.28–50 mg L⁻¹ in buffer samples. The assay was also implemented in human serum, and 0.2 and 0.7–25 mg L⁻¹ were the attained limit of detection and working range, respectively.     

Elimination of porosity in heavily rare-earth doped sol–gel derived silicate glass films

The combination of spin-coating and rapid thermal annealing is a very important sol–gel technique to prepare high quality silicate glass films, widely used in the fabrication of waveguides, photonic bandgap structures and other film-based optical devices. This work found that high rare-earth concentrations will seriously affect the optical quality of the films prepared by the spin-coating/rapid thermal annealing process, with pores with hundreds of nanometres in size being found in heavily rare-earth doped aluminosilicate glass films, causing significant light scattering. However, it was also found that a new recipe using acetylacetone was able to dramatically eliminate these pores and to improve the film optical quality, even for rare-earth concentrations as high as 15 mol%. This result will be useful for the fabrication of sol–gel derived devices based on rare-earth doped silicate glass films like active waveguides, functional films and photonic bandgap structures.     

Separation and detection of multiple pathogens in a food matrix by magnetic SERS nanoprobes

A rapid and sensitive method was developed here for separation and detection of multiple pathogens in food matrix by magnetic surface-enhanced Raman scattering (SERS) nanoprobes. Silica-coated magnetic probes (MNPs@SiO₂) of ∼100 nm in diameter were first prepared via the reverse microemulsion method using cetyltrimethylammonium bromide as a surfactant and tetraethyl orthosilicate as the silica precursor. The as-prepared MNPs@SiO₂ were functionalized with specific pathogen antibodies to first capture threat agents directly from a food matrix followed by detection using an optical approach enabled by SERS. In this scheme, pathogens were first immuno-magnetically captured with MNPs@SiO₂, and pathogen-specific SERS probes (gold nanoparticles integrated with a Raman reporter) were functionalized with corresponding antibodies to allow the formation of a sandwich assay to complete the sensor module for the detection of multiple pathogens in selected food matrices, just changing the kinds of Raman reporters on SERS probes. Here, up to two key pathogens, Salmonella enterica serovar Typhimurium and Staphylococcus aureus, were selected as a model to illustrate the probability of this scheme for multiple pathogens detection. The lowest cell concentration detected in spinach solution was 10³ CFU/mL. A blind test conducted in peanut butter validated the limit of detection as 10³ CFU/mL with high specificity, demonstrating the potential of this approach in complex matrices.     

Sedimentation measurements with the analytical ultracentrifuge with absorption optics: influence of Mie scattering and absorption of the particles

Analytical ultracentrifugation is one of the most powerful methods for the characterization of nanoparticles since the days of its invention due to its high resolution and statistical significance. Latexes with different sizes and their mixtures have been measured by sedimentation with the analytical ultracentrifuge (AUC) OPTIMA XL-I (Beckman Coulter, Palo Alto, CA, USA) with interference optics at λ ₀ = 675 nm and absorption optics at λ ₀ = 546 and 263 nm. Additionally, a blue pigment with high absorption characteristic at visible light has been investigated. A large influence of Mie scattering and Mie absorption on the particle size distribution with respect to absorption optics and samples with moderate or broad size distribution is observed. Therefore, the consideration of the Mie scattering effect is compulsory for most AUC measurements of nanoparticles with absorption optics.     

Development and characterization of an integrated silicon micro flow cytometer

This paper describes an innovative integrated micro flow cytometer that presents a new arrangement for the excitation/detection system. The sample liquid, containing the fluorescent marked particles/cells under analysis, is hydrodynamically squeezed into a narrow stream by two sheath flows so that the particles/cells flow individually through a detection region. The detection of the particles/cells emitted fluorescence is carried out by using a collection fiber placed orthogonally to the flow. The device is based on silicon hollow core antiresonant reflecting optical waveguides (ARROWs). ARROW geometry allows one to use the same channel to guide both the sample stream and the fluorescence excitation light, leading to a simplification of the optical configuration and to an increase of the signal-to-noise ratio. The integrated micro flow cytometer has been characterized by using biological samples marked with standard fluorochromes. The experimental investigation confirms the success of the proposed microdevice in the detection of cells. An erratum to this article can be found at     

Small-volume fiber-optic evanescent-wave absorption sensor for nitrite determination

A novel small-volume fiber-optic evanescent-wave absorption sensor based on the Griess–Ilosvay reaction has been developed and evaluated for nitrite determination. The sensor was constructed by inserting a decladded optical fiber into a transparent capillary to form an annular column microchannel. The Evanescent wave (EW) field produced on the optical fiber core surface penetrated into the surrounding medium and interacted with the azo dye, which was generated by the reaction of nitrite and nitrite-sensitive reagents. The detector was designed to be parallel to the axis of the optical fiber. The defined absorbance was linear with the concentration of nitrite in the range from 0.05 to 10 mg L⁻¹, and the detection limit was 0.02 mg L⁻¹ (3σ) with the relative standard deviation (RSD) of 2.6% (n = 8). The present sensor was successfully used to determine nitrite in real samples of mineral water, tap water, rain water, and seawater. The results were consistent with the data obtained by standard spectrophotometric method, showing potential of the proposed sensor for practical application.      

Enhancing the analytical performance of immunoassays that employ metal-enhanced fluorescence

In this work, we used a model assay system (polyclonal human IgG–goat antihuman IgG) to elucidate some of the key factors that influence the analytical performance of bioassays that employ metal-enhanced fluorescence (MEF) using silver nanoparticles (NPs). Cy5 dye was used as the fluorescent label, and results were compared with a standard assay performed in the absence of NPs. Two sizes of silver NPs were prepared with respective diameters of 60 ± 10 and 149 ± 16 nm. The absorption spectra of the NPs in solution were fitted accurately using Mie theory, and the dipole resonance of the 149-nm NPs in solution was found to match well with the absorption spectrum of Cy5. Such spectral matching is a key factor in optimizing MEF. NPs were deposited uniformly and reproducibly on polyelectrolyte-coated polystyrene substrates. Compared to the standard assay performed without the aid of NPs, significant improvements in sensitivity and in limit of detection (LOD) were obtained for the assay with the 149-nm NPs. An important observation was that the relative enhancement of fluorescence increased as the concentration of antigen increased. The metal-assisted assay data were analyzed using standard statistical methods and yielded a LOD of 0.086 ng/mL for the spectrally matched NPs compared to a value of 5.67 ng/mL obtained for the same assay in the absence of NPs. This improvement of ∼66× in LOD demonstrates the potential of metal-enhanced fluorescence for improving the analytical performance of bioassays when care is taken to optimize the key determining parameters.      

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.      

Magnetic particles functionalized with PAMAM-dendrimers and antibodies: a new system for an ELISA method able to detect Ara h3/4 peanut allergen in foods

An innovative enzyme-linked immunosorbent assay (ELISA) format based on antibody-coated magnetic micro-particles (MPs) for the sensitive detection of Ara h3/4 allergen in food is described. The immunosupport is suspended in the incubation solutions and the MPs with the captured allergen can be easily harvested on a magnet, separated from the solutions, and washed using an easy-to-use, fast and selective approach that allows its detection and quantification. Two differently coated MPs, ProteinA-Pn-b and MP-NH₂-PAMAM G 1.5 -Pn-b immunosupports, were tested. The functionalization of the MPs with PAMAM-sodium carboxylate dendrimers elicits a major stability on the immunoglobulin activity resulting in a threefold enhancement of the analytical sensitivity for the assay with respect to a ProteinA immobilization. Validation was carried out on two different matrices: corn flakes and biscuits. In the case of MP-NH₂-PAMAM G 1.5 -Pn-b immunosupport, limit of detection was found to be 0.2 mg peanuts/kg matrix in both matrices; the linear response range was demonstrated from 2.5 to 15 mg peanuts/kg matrix by performing statistical tests (homoscedasticity and Mandel fitting tests). Good accuracy and recovery (>80 ± 2%) were obtained. Different food samples were tested and the results were compared with those obtained with a commercially available ELISA kit. The results obtained in this work demonstrated the applicability of the immunomagnetic ELISA methods on real samples and the possibility to perform the assay with significantly reduced reagent and sample consumption.     

Simultaneous determination of Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol in oral fluid using isotope dilution liquid chromatography tandem mass spectrometry

The detection and confirmation of cannabinoids in oral fluid are important in forensic toxicology. Currently, the presence of Δ⁹-tetrahydrocannabinol (THC) is used for the detection of cannabis in oral fluid. A low concentration of 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH) is found in oral fluid, which suggested a convenient and low-sensitivity confirmation assay can be used in a routine forensic laboratory. In this study, a highly sensitive isotope dilution liquid chromatography–tandem mass spectrometry method following dansylation was successfully developed for simultaneous determination of THC and THC-COOH in oral fluid. The dansylated derivatives dramatically demonstrated and enhanced the sensitivity of THC and THC-COOH. To avoid signal influenced by the matrix, a 5-min liquid chromatography gradient program was evaluated and optimized, which reduced the sample diffusion and caused sharp peaks (less than 12 s) and thus helped to achieve detection at a low level. The sensitivity, accuracy, and precision were also evaluated, and high quantitative accuracy and precision were obtained. The limit of quantitation of this approach was 25 pg/mL for THC and 10 pg/mL for THC-COOH in oral fluid. Finally, the method was successfully applied to eight suspected cannabis users. Among them, in six oral fluid samples THC-COOH was determined at a concentration from 13.1 to 47.2 pg/mL.     

Thiol-stabilized luminescent CdTe quantum dot as biological fluorescent probe for sensitive detection of methyl parathion by a fluoroimmunochromatographic technique

Quantum dots (QDs) are preferred as high-resolution biological fluorescent probes because of their inherent optical properties compared with organic dyes. This intrinsic property of QDs has been made use of for sensitive detection of methylparathion (MP) at picogramme levels. The specificity of the assay was attributed to highly specific immunological reactions. Competitive binding between free MP and CdTe QD bioconjugated MP (MP-BSA-CdTe) with immobilized anti-MP IgY antibodies was monitored in a flow-injection system. The fluorescence intensity of MP-BSA-CdTe bioconjugate eluted from the column was found to be directly proportional to the free MP concentration. Hence, it was possible to detect MP in a linear range of 0.1–1 ng mL⁻¹ with a regression coefficient R ² = 0.9905. In this investigation, IgY proved advantageous over IgG class immunoglobulins in terms of yield, stability, cost effectiveness, and enhancement of assay sensitivity. The photo-absorption spectrum of bioconjugated CdTe QD (λ _max = 310 nm) confirmed nano-biomolecular interactions. The results suggest the potential application of bioconjugation and nano-biomolecular interactions of QDs for biological labeling and target analyte detection with high sensitivity.     

Qualitative detection of desmopressin in plasma by liquid chromatography–tandem mass spectrometry

This work describes a liquid chromatography–electrospray tandem mass spectrometry method for detection of desmopressin in human plasma in the low femtomolar range. Desmopressin is a synthetic analogue of the antidiuretic hormone arginine vasopressin and it might be used by athletes as a masking agent in the framework of blood passport controls. Therefore, it was recently added by the World Anti-Doping Agency to the list of prohibited substances in sport as a masking agent. Mass spectrometry characterization of desmopressin was performed with a high-resolution Orbitrap-based mass spectrometer. Detection of the peptide in the biological matrix was achieved using a triple-quadrupole instrument with an electrospray ionization interface after protein precipitation, weak cation solid-phase extraction and high performance liquid chromatography separation with an octadecyl reverse-phase column. Identification of desmopressin was performed using three product ions, m/z 328.0, m/z 120.0, and m/z 214.0, from the parent ion, m/z 535.5. The extraction efficiency of the method at the limit of detection was estimated as 40% (n = 10), the ion suppression as 5% (n = 10), and the limit of detection was 50 pg/ml (signal-to-noise ratio greater than 3). The selectivity of the method was verified against several endogenous and synthetic desmopressin-related peptides. The performance and the applicability of the method were tested by analysis of clinical samples after administration of desmopressin via intravenous, oral, and intranasal routes. Only after intravenous administration could desmopressin be successfully detected.     

Serum-based ALYGNSA immunoassay for the prostate cancer biomarker, total prostate-specific antigen (tPSA)

Prostate-specific antigen (PSA) is a serum glycoprotein overproduced by the prostate in prostate cancer (≥4 ng/mL in the bloodstream). An immunoassay for total PSA (tPSA) was developed using the ALYGNSA method to enhance capture antibody orientation and a limit of detection of 0.63 ng/mL was reported, a limit 15-fold lower than a commercial tPSA ELISA assay. This ALYGNSA assay, however, was performed using only buffer-based proteins and blocking agents (Mackness et al., Anal Bioanal Chem 396:681–686, 2010). To improve the clinical application of this system, a serum-based tPSA ALYGNSA was developed employing human serum. This assay also resulted in a limit of detection of 0.63 ng/mL of tPSA protein. The findings reported here provide support for the clinical application of this assay for diagnosis, progression, treatment, and possible recurrence of prostate cancer.     

A hard microflow cytometer using groove-generated sheath flow for multiplexed bead and cell assays

With a view toward developing a rugged microflow cytometer, a sheath flow system was micromachined in hard plastic (polymethylmethacrylate) for analysis of particles and cells using optical detection. Six optical fibers were incorporated into the interrogation region of the chip, in which hydrodynamic focusing narrowed the core stream to ∼35 μm × 40 μm. The use of a relatively large channel at the inlet as well as in the interrogation region (375 μm × 125 μm) successfully minimized the risk of clogging. The device could withstand pressures greater than 100 psi without leaking. Assays using both coded microparticles and cells were demonstrated using the microflow cytometer. Multiplexed immunoassays detected nine different bacteria and toxins using a single mixture of coded microspheres. A549 cancer cells processed with locked nucleic acid probes were evaluated using fluorescence in situ hybridization.     

On-line monitoring of gas-phase bioreactors for biogas treatment: hydrogen sulfide and sulfide analysis by automated flow systems

Biogas is produced by biological processes under anaerobic conditions and may contain up to 20,000 ppm_v hydrogen sulfide (H₂S), a corrosive substance that attacks power engines and can affect the health of the industrial staff. H₂S must be removed from the biogas, especially in co-generation facilities where the biogas is burnt for energy production. Nowadays, biofiltration is being studied and considered as an interesting alternative for removing H₂S from the biogas besides classical chemical processes. The novelty of this work is the design and construction of an automated H₂S on-line analyser to assess the composition of the liquid and gas phases of gas-phase bioreactors. The analyser is made of two parallel flow configurations which share the same detection device. The first configuration is a single-channel flow injection analyser (FIA) to detect S²⁻ in the liquid phase. The second configuration is a continuous flow analyser (CFA) with a gaseous diffusion step (GD–CFA) for detecting H₂S in the gas phase. The diffusion step enables separation of the H₂S_(g) from the sample and its conversion into a detectable chemical species (S²⁻). S²⁻ detection was performed with an Ag₂S ion-selective electrode (ISE) selective to . The main response parameters of the FIA system are a linear range between 3 × 10⁻⁵ and 1 × 10⁻¹ mol L⁻¹ S²⁻ (0.61–3,200 mg L⁻¹), with a sensitivity of 27.9 mV decade⁻¹ and a detection limit of 1.93 × 10⁻⁵ mol L⁻¹ S²⁻. The GD–CFA configuration presents a linear range between 400 and 10,000 ppm_v H₂S_(g) with a sensitivity of 26.1 mV decade⁻¹ and a detection limit of 245 ppm_v H₂S. The proposed analyser was used by analysing real gas and liquid samples with optimal results at a full-scale biotrickling filter for biogas treatment at a municipal wastewater treatment plant. Figure The novelty of this work is the design and construction of an automated H₂S on-line analyzer to assess the composition of the liquid and gas phases of gas-phase bioreactors. The analyser is made of two parallel flow configurations which share the same detection device. The first configuration is a single-channel flow injection analyser (FIA) to detect S^2- in the liquid phase. The second configuration is a continuous flow analyser (CFA) with a gaseous diffusion step (GD-CFA) for detecting H₂S in the gas phase.