Optical sensing in microfluidic lab-on-a-chip by femtosecond-laser-written waveguides

We use direct femtosecond laser writing to integrate optical waveguides into a commercial fused silica capillary electrophoresis chip. High-quality waveguides crossing the microfluidic channels are fabricated and used to optically address, with high spatial selectivity, their content. Fluorescence from the optically excited volume is efficiently collected at a 90° angle by a high numerical aperture fiber, resulting in a highly compact and portable device. To test the platform we performed electrophoresis and detection of a 23-mer oligonucleotide plug. Our approach is quite powerful because it allows the integration of photonic functionalities, by simple post-processing, into commercial LOCs fabricated with standard techniques. Figure Femtosecond laser written waveguides can selectively excite fluorescence in a microfluidic channel of a commercial lab-on-a-chip. A compact scheme for on-chip detection by laser induced fluorescence is applied to capillary electrophoresis of a 23-mer Cy3-labeled oligonucleotide     

Impedimetric genosensors for the detection of DNA hybridization

Impedance spectroscopy is proposed as the transduction principle for detecting the hybridization of DNA complementary strands. In our experiments, different DNA oligonucleotides were used as model gene substances. The gene probe is first immobilized on a graphite-epoxy composite working electrode based genosensor. Detection principle is based on changes of impedance spectra of a redox marker, the ferro/ferricyanide couple, after hybridization with target DNA. Resistance offered to the electrochemical reaction serves as the working signal, allowing for an unlabelled gene assay.      

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     

Cation identity dependence of crown ether photonic crystal Pb<Superscript>2+</Superscript> sensing

We quantitatively modeled the volume phase transition of a hydrogel containing a crystalline colloidal array with a crown ether ligand which binds Pb²⁺. The hydrogel volume response and the wavelength diffracted depend on the Pb²⁺ concentration and on both the ionic strength and the valence of the nonbinding ionic species. We successfully modeled the response of this hydrogel Pb²⁺ sensor to ionic strength and the cation valence of the added salts. Figure Cation identity dependence of crown ether photonic crystal Pb⁺ sensing     

Iron fractionation for corrosion products from natural gas pipelines by continuous-flow sequential extraction

The forms and quantities of iron species in corrosion product samples from natural gas pipelines were examined, using a continuous-flow sequential extraction system. Sequential extraction consists of four steps that dissolve water soluble iron (FeSO₄), acid soluble iron (FeCO₃), reducible iron (Fe-(oxyhydr)oxides) and oxidisable iron (FeS₂) fractions, respectively. Selectivity of extracting reagents for particular iron species was evaluated by determination of co-extracted anions, using ion chromatography, and evolved CO₂, using indirect flame atomic absorption spectrometer (FAAS). Iron was found predominantly in the reducible fraction (61–99%), indicating that Fe-(oxyhydr)oxides are the major constituents of the corrosion products.      

SPME in environmental analysis

Recent advances in the use of solid-phase microextraction (SPME) in environmental analysis, including fiber coatings, derivatization techniques, and in-tube SPME, are reviewed in this article. Several calibration methods for SPME, including traditional calibration methods, the equilibrium extraction method, the exhaustive extraction method, and several diffusion-based calibration methods, are presented. Recent developed SPME devices for on-site sampling and several applications of SPME in environmental analysis are also introduced.      

Novel coumarin-based fluorescent pH indicators,probes and membranes covering a broad pH range

A new family of coumarin-based pH indicators was synthesized. They are sensitive to pH in either weakly acidic or weakly basic solution. The indicators possess moderate to high brightness, excellent photostability and compatibility with light-emitting diodes. The indicators were covalently immobilized on the surface of amino-modified polymer microbeads which in turn were incorporated into a hydrogel matrix to obtain novel pH-sensitive materials. When a mixture of two different microbeads is used, the membranes are capable of optical pH sensing over a very wide range comparable to the dynamic range of the glass electrode (pH 1–11). A new family of coumarin-based pH indicators is synthesized for the use in either weakly acidic or weakly basic solution. The indicators possess moderate to high brightness, excellent photostability and compatibility with light-emitting diodes. Novel pH-sensitive materials are obtained by covalent immobilization of the indicators on the surface of amino-modified polymer microbeads which in turn are incorporated into a hydrogel matrix. Sensing of pH over a very wide range also becomes possible.     

Chemiluminescent detection of DNA hybridization using gold nanoparticles as labels

A chemiluminescent (CL) detection method has been developed for DNA hybridization. The assay relies on a sandwich-type DNA hybridization in which gold nanoparticles modified with alkylthiol-capped oligonucleotide strands are used as probes to monitor the presence of the specific target DNA. The , which is the dissolving product of the gold nanoparticles anchored on the DNA hybrids, serves as an analyte in the H₂O₂–luminol– CL reaction for the indirect measurement of the target DNA. The combination of the remarkable sensitivity of the CL analysis with the large number of released from each DNA hybrid allows a detection limit at levels as low as 0.1 pM of the target DNA. Moreover, with a further silver amplification step, the detection limit will be pushed down to the femtomolar domain.      

Integration of microcolumns and microfluidic fractionators on multitasking centrifugal microfluidic platforms for the analysis of biomolecules

This work demonstrates the development of microfluidic compact discs (CDs) for protein purification and fractionation integrating a series of microfluidic features, such as microreservoirs, microchannels, and microfluidic fractionators. The CDs were fabricated with polydimethylsiloxane (PDMS), and each device contained multiple identical microfluidic patterns. Each pattern employed a microfluidic fractionation feature with operation that was based on the redirection of fluid into an isolation chamber as a result of an overflow. This feature offers the advantage of automated operation without the need for any external manipulation, which is independent of the size and the charge of the fractionated molecules. The performance of the microfluidic fractionator was evaluated by its integration into a protein purification microfluidic architecture. The microfluidic architecture employed a microchamber that accommodated a monolithic microcolumn, the fractionator, and an isolation chamber, which was also utilized for the optical detection of the purified protein. The monolithic microcolumn was polymerized “in situ” on the CD from a monolith precursor solution by microwave-initiated polymerization. This technique enabled the fast, efficient, and simultaneous polymerization of monoliths on disposable CD microfluidic platforms. The design of the CD employed allows the integration of various processes on a single microfluidic device, including protein purification, fractionation, isolation, and detection.      

New near-infrared LIBS detection technique for sulfur

Sulfur has been detected in a spectral window (around 868 nm) previously unexplored by laser-induced breakdown spectrometry (LIBS), using an ablation laser with an ultraviolet wavelength, a gated detector, and inert ambient gas at a low, controlled pressure. This spectral window enables new-generation gated iCCD cameras to be used, which have adequate quantum efficiencies up to 900 nm. Application of our technique can substantially improve signal strength and thus extends the ability of LIBS to detect many nonmetallic elements.      

Development and field evaluation of a new diffusive sampler for Hydrogen Sulphide in the ambient air

A diffusive sampler for the determination of hydrogen sulphide (H₂S) based on collection on a paper filter coated with silver nitrate followed by optical densitometric determination of the metal sulphide was developed. Laboratory tests were conducted in controlled atmosphere to evaluate linearity, uptake rate, face velocity effects, sample stability, influence of relative humidity and of interferents, precision and accuracy. The measured uptake rate for H₂S was determined in experiments involving sampling at different concentration levels in comparison to a wet standard colorimetric technique. The precision of the measurements for co-located passive samplers was lower than 15%. The accuracy of the data collected is within 20% of the actual value measured by the wet method. The sampler is capable of reliable measurements of H₂S at common levels of a polluted atmosphere in urban settings yielding average concentration levels over one month and beyond. Diffusive sampling can be adopted to analyse in detail the temporal and spatial trends of H₂S concentration in ambient air and in specific historic buildings or in museums. Figure At the end of sampling cap #2 is removed and optical density is measured     

Detection of bacteria by surface-enhanced Raman spectroscopy

The detection and identification of dilute bacterial samples by surface-enhanced Raman spectroscopy has been explored by mixing aqueous suspensions of bacteria with a suspension of nanocolloidal silver particles. An estimate of the detection limit of E. coli was obtained by varying the concentration of bacteria. By correcting the Raman spectra for the broad librational OH band of water, reproducible spectra were obtained for E. coli concentrations as low as approximately 10³ cfu/mL. To aid in the assignment of Raman bands, spectra for E. coli in D₂O are also reported. Figure Light scattering apparatus used to detect bacteria     

The use of electrochemical impedance spectroscopy for biosensing

This review introduces the basic concepts and terms associated with impedance and techniques of measuring impedance. The focus of this review is on the application of this transduction method for sensing purposes. Examples of its use in combination with enzymes, antibodies, DNA and with cells will be described. Important fields of application include immune and nucleic acid analysis. Special attention is devoted to the various electrode design and amplification schemes developed for sensitivity enhancement. Electrolyte insulator semiconductor (EIS) structures will be treated separately. Figure An alternating current which is forced to pass an interface is sensitive to surface changes and will detect impedance changes due to biomolecule immobilisation or formation of a recognition complex. This can be used for the construction of biosensor electrodes     

Detection and identification of IHN and ISA viruses by isothermal DNA amplification in microcapillary tubes

Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV or ISAV were recognized by MPPs. Circularized MPPs were then captured on the inner surfaces of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA produced DNA concatamers, which were labeled with fluorescent SYBR Green II nucleic acid stain, and measured by microfluorimetry. Molecular padlock probes, combined with this method of surface-associated isothermal RCA, exhibited high selectivity without the need for thermal cycling. This method is applicable to the design of low-power field sensors capable of multiplex detection of viral, bacterial, and protozoan pathogens within localized regions of microcapillary tubes.      

Different iron-chelating properties of pyochelin diastereoisomers revealed by LC/MS

Pyochelin is a siderophore and virulence factor common to Burkholderia cepacia and several Pseudomonas strains. It is isolated from bacterial media as a mixture of two epimers, which readily equilibrate in most solvents. Experiments based on high-performance liquid chromatography/electrospray ionization mass spectrometry are reported here, allowing the investigation of the different Fe(III)-chelating properties of pyochelin diastereomers in solution without the need for labourious isolation. It is demonstrated in this study that only one of the two pyochelin diastereomers is able to chelate Fe(III); no Fe(III) complexes of the other diastereomer could be detected. The Fe(III)–pyochelin complex exhibited a 1:1 metal-to-siderophore ratio and no evidence for other stoichiometries was found.      

Capillary HPLC–ICP MS mapping of selenocompounds in spots obtained from the 2-D gel electrophoresis of the water-soluble protein fraction of selenized yeast

A method based on ICP collision-cell MS detection in capillary HPLC was developed to gain an insight into the purity and identity of selenium-containing proteins separated by 1-D and 2-D electrophoresis. The bands and spots obtained after the separation of water-soluble proteins in selenized yeast were digested with trypsin prior to chromatography. Selenium could be detected down to the subpicogram level. The method, assisted by information obtained by MALDI TOF MS on the 5000 Da cut-off fraction, permitted the purity of bands and spots to be estimated and the efficiency of tryptic digestion and the quantity of selenium present in individual peptides to be evaluated. Owing to the high sensitivity and the lack of matrix suppression effects, the method provided chromatograms with signal-to-noise ratios of 10–1000 in conditions where the common ES Q–TOF MS detection failed.      

Rapid protein expression analysis with an interferometric biosensor for monitoring protein production

The concentration of a recombinantly expressed protein has to be monitored to select optimal expression conditions throughout the protein production process. Today this is usually achieved semiquantitatively with sodium dodecyl sulfate polyacrylamide gel electrophoresis/western blotting or with ELISAs, which are time- and labor-intensive methods. In this paper the applicability of a label-free sensor system based on a Young interferometer is presented as an alternative for the monitoring of recombinant protein production. Once a protein is successfully produced, the interferometric biosensor allows any protein–protein interaction to be characterized in a label-free manner. This is demonstrated with an antibody/antigen pair, where the antibody is directed against a four-amino-acid tag used for protein expression analysis as well as purification during recombinant protein production. Label-free detection of the tagged protein is shown both in buffer and in bacterial cell lysate as a sample matrix. The system exhibiting a low limit of detection, low drift and reliable operation is compared with a commercial surface plasmon resonance sensor and a competitive ELISA. Figure 1 Waveguide sensor chip; grating (green) illuminated by a red light source. Image courtesy of Unaxis Optics Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus

We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA analysis. The PDA chip comprises an 8 × 6 array of photodiodes each with a diameter of 600 μm. Each photodiode element acts both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction (PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a broader range of target DNA concentration by controlling the silver development time. Figure An optical image of the PDA chip and target DNA detection through silver enhancement Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Photo-induced cold vapor generation with low molecular weight alcohol, aldehyde, or carboxylic acid for atomic fluorescence spectrometric determination of mercury

With UV irradiation, Hg²⁺ in aqueous solution can be converted into Hg⁰ cold vapor by low molecular weight alcohols, aldehydes, or carboxylic acids, e.g., methanol, formaldehyde, acetaldehyde, glycol, 1,2-propanediol, glycerol, acetic acid, oxalic acid, or malonic acid. It was found that the presence of nano-TiO₂ more or less improved the efficiency of the photo-induced chemical/cold vapor generation (photo-CVG) with most of the organic reductants. The nano-TiO₂-enhanced photo-CVG systems can be coupled to various analytical atomic spectrometric techniques for the determination of ultratrace mercury. In this work, we evaluated the application of this method to the atomic fluorescence spectrometric (AFS) determination of mercury in cold vapor mode. Under the optimized experimental conditions, the instrumental limits of detection (based on three times the standard deviation of 11 measurements of a blank solution) were around 0.02–0.04 μg L⁻¹, with linear dynamic ranges up to 15 μg L⁻¹. The interference of transition metals and the mechanism of the photo-CVG are briefly discussed. Real sample analysis using the photo-CVG-AFS method revealed that it was promising for water and geological analysis of ultralow levels of mercury. Image of the photo-CVG instrumentation showing the photoreactor inside the water cooling unit