Optical biosensors based on integrated polymer light source and polymer photodiode

Evanescent coupling is used to couple light from an organic Lambertian emitter into a single‐mode planar waveguide. A polymer light emitting diode pumps a photoluminescent layer located directly on top of the waveguide. At the out‐coupling grating stage, a fully organic mini‐spectrometer compatible with monolithical integration on optical bio chips has been developed. It consists of a single‐mode waveguide with integrated diffraction grating and a dense array of polymer photodiodes as sensing element. An overall spectral resolution of down to 5 nm has been achieved with the integrated optoelectronic system. As a proof of principle the fully organic optical device has been used in combination with a fluidic system to demonstrate an absorption‐based bio‐test with mouse immunoglobulin G. In a further step towards low‐cost and disposable lab‐on‐chip biosensors, the mentioned organic building blocks have been combined with a surface plasmon stack integrated directly onto the single mode waveguide. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2010     

Towards microalbuminuria determination on a disposable diagnostic microchip with integrated fluorescence detection based on thin-film organic light emitting diodes

As a first step towards a fully disposable stand-alone diagnostic microchip for determination of urinary human serum albumin (HSA), we report the use of a thin-film organic light emitting diode (OLED) as an excitation source for microscale fluorescence detection. The OLED has a peak emission wavelength of 540 nm, is simple to fabricate on flexible or rigid substrates, and operates at drive voltages below 10 V. In a fluorescence assay, HSA is reacted with Albumin Blue 580, generating a strong emission at 620 nm when excited with the OLED. Filter-less discrimination between excitation light and generated fluorescence is achieved through an orthogonal detection geometry. When the assay is performed in 800 microm deep and 800 microm wide microchannels on a poly(dimethylsiloxane)(PDMS) microchip at flow rates of 20 microL min(-1), HSA concentrations down to 10 mg L(-1) can be detected with a linear range from 10 to 100 mg L(-1). This sensitivity is sufficient for the determination of microalbuminuria (MAU), an increased urinary albumin excretion indicative of renal disease (clinical cut-off levels: 15-40 mg L(-1)).     

Spectrum of light scattered from polydisperse polymer solutions

We examine several methods for analyzing the spectrum of light scattered from polydisperse polymer solutions. General expressions are reviewed for the inelastic scattering spectra and integrated intensities due to the pure translational normal mode of motion, using both heterodyne and homodyne detection, in terms of the molecular weights, concentrations, scattering form factors, and diffusion coefficients of the individual polymeric species, These results are used to obtain general expressions for the limiting slopes and intercepts of various rearranged versions of the equation I(v) = (A/π)²/(v² + γ²) that permit linear plotting: I(v) is the intensity of light scattered at frequency v, A is the integrated intensity, and γ is the spectral halfwidth, K²D/2π, where K is the scattering vector and D the diffusion coefficient. These results are applied to the special case of a Schulz-Zimm distribution, neglecting form factors, to obtain explicit expressions relating the average diffusion coefficients determined by these procedures to other measurable quantities: the mean polymer radius; the diffusion coefficient of the weight-average species; and, together with the weight-average sedimentation coefficient, the weight-average molecular weight. Numerical calculations for two particular cases indicate the relative merits of the various data analysis procedures. Homodyne detection gives average values that are closer to weight averages than does heterodyne detection.     

Towards a portable microchip system with integrated thermal control and polymer waveguides for real-time PCR

A novel real-time PCR microchip platform with integrated thermal system and polymer waveguides has been developed. The integrated polymer optical system for real-time monitoring of PCR was fabricated in the same SU-8 layer as the PCR chamber, without additional masking steps. Two suitable DNA binding dyes, SYTOX Orange and TO-PRO-3, were selected and tested for the real-time PCR processes. As a model, cadF gene of Campylobacter jejuni has been amplified on the microchip. Using the integrated optical system of the real-time PCR microchip, the measured cycle threshold values of the real-time PCR performed with a dilution series of C. jejuni DNA template (2 to 200 pg/microL) could be quantitatively detected and compared with a conventional post-PCR analysis (DNA gel electrophoresis). The presented approach provided reliable real-time quantitative information of the PCR amplification of the targeted gene. With the integrated optical system, the reaction dynamics at any location inside the micro reaction chamber can easily be monitored.     

Simultaneous counting of two subsets of leukocytes using fluorescent silica nanoparticles in a sheathless microchip flow cytometer

A portable flow cytometer has been recognized as an important tool for many clinical applications such as HIV/AIDS screening in developing countries and regions with limited medical facilities and resources. Conventional flow cytometers typically require multiple detectors for simultaneous identification of multiple subsets of immune cell. To minimize the number of detectors toward portable flow cytometry or to analyze multi-parametric cellular information with minimum number of detectors in conventional flow cytometers, we propose a versatile multiplexed cell-counting method using functional silica nanoparticles (SiNPs). FITC-doped SiNPs, which are 100 times brighter than the FITC molecules itself, were used as new intensity-based fluorescent dye complexes to simultaneously measure two subsets of leukocytes using a single detector. CD45(+)CD4(+) cells tagged with these FITC-doped SiNPs were 50 times brighter than CD45(+)CD4(-) cells tagged only with FITC. To make the overall system compact, a disposable microchip flow cytometer that does not require sheath flow was developed. Combining these dye-doped SiNPs based detection schemes and the sheathless microchip flow cytometer scheme, we successfully identified and counted two subsets of leukocytes simultaneously (R(2) = 0.876). These approaches can be the building blocks for a truly portable and disposable flow cytometer for various clinical cytometry applications.     

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     

Microfluidic flow rate detection based on integrated optical fiber cantilever

We demonstrate an integrated microfluidic flow sensor with ultra-wide dynamic range, suitable for high throughput applications such as flow cytometry and particle sorting/counting. A fiber-tip cantilever transduces flow rates to optical signal readout, and we demonstrate a dynamic range from 0 to 1500 microL min(-1) for operation in water. Fiber-optic sensor alignment is guided by preformed microfluidic channels, and the dynamic range can be adjusted in a one-step chemical etch. An overall non-linear response is attributed to the far-field angular distribution of single-mode fiber output.     

Sample pretreatment on a microchip with an integrated electrospray emitter

This study presents a microbead-packed PDMS microchip with an integrated electrospray emitter for sample pretreatment prior to sheathless ESI-MS. We prove the concept of analytical functions integrated onto a cm-sized area of a single bulk material. The microchip consists of two PDMS substrates replicated from SU-8 fabricated silicon wafer masters, bonded together after oxidation by corona discharge treatment. The channel within the microchip contains a grid structure that was used to trap 5 microm hypercross-linked polystyrene beads. The beads acted as a medium for sample desalting and enrichment. Electrical contact for the sheathless ESI process was achieved by coating the integrated emitter with conductive graphite powder after applying a thin layer of PDMS as glue. The coating as well as the bond of the PDMS structures showed excellent durability. A continuous spray was obtained from the microchip for over 800 h in a long-term electrospray stability experiment. Desalting and enrichment of neuropeptides from a physiological salt solution was successful by loading the sample onto the packed beads, followed by a washing and an eluting step. The results were obtained and evaluated using a TOF MS. An LOD of approximately 20 fmol (loaded onto the beads) for angiotensin II was obtained from a sample of neuropeptides dissolved in physiological salt solution.     

Counting of Escherichia coli by a microflow cytometer based on a photonic–microfluidic integrated device

Counting of Escherichia coli DH5α‐cell suspensions in PBS is performed using a microflow cytometer based on a photonic–microfluidic integrated device. Side‐scattered light signals are used to count the E. coli cells. A detection efficiency of 92% is achieved when compared with the expected count from a hemocytometer. The detection efficiency is correlated to the ratio of sample to sheath flow rates. It is demonstrated that E. coli can be easily distinguished from beads of similar sizes (2–4 μm) as their scattering intensities are different.     

Off-line form of the Michaelis-Menten equation for studying the reaction kinetics in a polymer microchip integrated with enzyme microreactor

We firstly transformed the traditional Michaelis-Menten equation into an off-line form which can be used for evaluating the Michaelis-Menten constant after the enzymatic reaction. For experimental estimation of the kinetics of enzymatic reactions, we have developed a facile and effective method by integrating an enzyme microreactor into direct-printing polymer microchips. Strong nonspecific adsorption of proteins was utilized to effectively immobilize enzymes onto the microchannel wall, forming the integrated on-column enzyme microreactor in a microchip. The properties of the integrated enzyme microreactor were evaluated by using the enzymatic reaction of glucose oxidase (GOx) with its substrate glucose as a model system. The reaction product, hydrogen peroxide, was electrochemically (EC) analyzed using a Pt microelectrode. The data for enzyme kinetics using our off-line form of the Michaelis-Menten equation was obtained (K(m) = 2.64 mM), which is much smaller than that reported in solution (K(m) = 6.0 mM). Due to the hydrophobic property and the native mesoscopic structure of the poly(ethylene terephthalate) film, the immobilized enzyme in the microreactor shows good stability and bioactivity under the flowing conditions.     

Fabrication of SU-8 based microchip electrophoresis with integrated electrochemical detection for neurotransmitters

A new SU-8 based microchip capillary electrophoresis (MCE) device has been developed for the first time with integrated electrochemical detection. Embedded electrophoretic microchannels have been fabricated with a multilayer technology based on bonding and releasing steps of stacked SU-8 films. This technology has allowed the monolithic integration in the device of the electrochemical detection system based on platinum electrodes. The fabrication of the chips presented in this work is totally compatible with reel-to-reel techniques, which guarantee a low cost and high reliability production. The influence of relevant experimental variables, such as the separation voltage and detection potential, has been studied on the SU-8 microchip with an attractive analytical performance. Thus, the effective electrical isolation of the end-channel amperometric detector has been also demonstrated. The good performance of the SU-8 device has been proven for separation and detection of the neurotransmitters, dopamine (DA) and epinephrine (EP). High efficiency (30,000-80,000 N/m), excellent precision, good detection limit (450 nM) and resolution (0.90-1.30) has been achieved on the SU-8 microchip. These SU-8 devices have shown a better performance than commercial Topas (thermoplastic olefin polymer of amorphous structure) microchips. The low cost and versatile SU-8 microchip with integrated platinum film electrochemical detector holds great promise for high-volume production of disposable microfluidic analytical devices.     

Integrated multilayer flow system on a microchip.

We utilized microchip technology and found that the multilayer flow of liquids can be formed in microchannels. Liquid/liquid interfaces were formed parallel to the side wall of the microchannels, because the surface tension and friction force are stronger than the force of gravity. A water/ethylacetate/water interface was formed in a 70-microm-wide and 30-microm-deep channel. The interface was observed to be quite stable and to be maintained for a distance of more than 18 cm. As an example of a multilayer flow application, we demonstrated the liquid/liquid extraction of Co-dimethylaminophenol complex in a microchannel. The solvent-extraction process of the complex into m-xylene in the multilayer flow was found to reach equilibrium in 4 s, while it took 60 s in a simple two-phase extraction.     

Coupling microchip electrophoresis with MALDI-TOF-MS based on a freezing technique

A freezing technique protocol was proposed for coupling microchip electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALD1-TOF-MS).The microfluidic flow was frozen immediately after electrophoresis on microfluidic chip and the separated analyte molecules were kept in their zone pattern in the electrophoresis.Then,the frozen-chip was lyophilized and sent into TOF-MS instrument as a MALDI target,and the analyte molecules in the microfluidic channels were subjected to analysis by mass spectrometry.This approach could eliminate sample cross-contamination, providing a new interface for microchip electrophoresis and MALDI-MS.     

Temperature behaviour of a liquid crystal comb polymer: Light scattering and noise of the scattered light

Measurements of the intensity of the monochromatic light transmitted through and scattered by a comb polymer with a polyacrylamide main chain were performed between room temperature and the isotropization temperature of the polymer. The stationary noise of the light scattered at low angle was measured in the same temperature interval. The transmitted intensity is observed to increase strongly above the smectic S_I2-S_c2 transition, where the intensity of the light scattered at low angles is maximized. The power dissipated by the molecular fluctuations dramatically increases above the transition between the two smectic phases. The spectral density curves display a Lorentzian character only below the S_I2-S_c2 transition. At higher temperatures, a more complex frequency behaviour of the stationary noise spectra is observed. Such a behaviour is interpreted in terms of a model explicitly invoking the effect of the Brownian movement of segments of the main chain (backbone) of the polymer on the side chain fluctuations. The parameters governing the Brownian movements of both main and side chains, and their evolution with temperature, are determined and discussed in the light of a simple structural model.     

Capillary electrophoresis coupled to mass spectrometry from a polymer modified poly(dimethylsiloxane) microchip with an integrated graphite electrospray tip

Hybrid capillary-poly(dimethysiloxane)(PDMS) microchips with integrated electrospray ionization (ESI) tips were directly fabricated by casting PDMS in a mould. The shapes of the emitter tips were drilled into the mould, which produced highly reproducible three-dimensional tips. Due to the fabrication method of the microfluidic devices, no sealing was necessary and it was possible to produce a perfect channel modified by PolyE-323, an aliphatic polyamine coating agent. A variety of different coating procedures were also evaluated for the outside of the emitter tip. Dusting graphite on a thin unpolymerised PDMS layer followed by polymerisation was proven to be the most suitable procedure. The emitter tips showed excellent electrochemical properties and durabilities. The coating of the emitter was eventually passivated, but not lost, and could be regenerated by electrochemical means. The excellent electrochemical stability was further confirmed in long term electrospray experiments, in which the emitter sprayed continuously for more than 180 h. The PolyE-323 was found suitable for systems that integrate rigid fused silica and soft PDMS technology, since it simply could be applied successfully to both materials. The spray stability was confirmed from the recording of a total ion chromatogram in which the electrospray current exhibited a relative standard deviation of 3.9% for a 30 min run. CE-ESI-MS separations of peptides were carried out within 2 min using the hybrid PDMS chip resulting in similar efficiencies as for fused silica capillaries of the same length and thus with no measurable band broadening effects, originating from the PDMS emitter.     

Use of a thermal lens microscope in integrated catecholamine determination on a microchip

A new method for determination for catecholamines (CA) utilizing microchip technology and a thermal lens microscope has been developed. Microchannels with a 250 microm x 10 microm cross section were used for mixing, reaction, and detection. Epinephrine (EP), nor-epinephrine (NE), dopamine (DA), and L-dopa (LD) were determined by using coloring oxidization to aminochromes by sodium metaperiodate. A thermal lens microscope (TLM) was used for detection of the product. The sensitivity of the system was comparable for the four CA and required only 15 s for mixing of sample and reagent. The calibration lines indicated excellent linearity for concentrations of 5-20 microg mL(-1). The relative standard deviations for 10 microg mL(-1) solution were 1.08, 2.18, 2.2, and 2.5% for EP, NE, DA, and LD, respectively. CA in pharmaceutical injections were also determined by use of the system and the results correlated very well with nominal values. Results obtained by use of the integrated system suggested there was a sufficient possibility to realize high-throughput medical diagnosis systems.     

Simultaneous high speed optical and impedance analysis of single particles with a microfluidic cytometer

We describe a microfluidic cytometer that performs simultaneous optical and electrical characterisation of particles. The microfluidic chip measures side scattered light, signal extinction and fluorescence using integrated optical fibres coupled to photomultiplier tubes. The channel is 80 μm high and 200 μm wide, and made from SU-8 patterned and sandwiched between glass substrates. Particles were focused into the analysis region using 1-D hydrodynamic focusing and typical particle velocities were 0.1 ms(-1). Excitation light is coupled into the detection channel with an optical fibre and focused into the channel using an integrated compound air lens. The electrical impedance of particles is measured at 1 MHz using micro-electrodes fabricated on the channel top and bottom. This data is used to accurately size the particles. The system is characterised using a range of different sized polystyrene beads (fluorescent and non-fluorescent). Single and mixed populations of beads were measured and the data compared with a conventional flow cytometer.     

Liquid crystal comb polymer with polar mesogenic and aliphatic side groups II. Noise of the scattered light

Measurements of static light scattering and of the stationary noise of the scattered light have been performed as functions of temperature on the comb polymer whose synthesis and static structural properties are discussed in the preceding paper, Part I. The intrinsic optical noise strongly increases when the nematic phase is entered. An additional maximum in the noise, observed at lower temperatures, is attributed to a rearrangement of optical domains, introducing boundary regions where the fluctuations of loosely packed side groups are enhanced. The spectral density of the observed noise is definitely non-Lorentzian and can be fitted by the superposition of two pure Lorentzian components with different cut-off frequencies, attributed to the statistically independent random motion of mesogenic and aliphatic side chains. The noise level is reduced at all temperatures by an applied electrical field; in particular, the field is effective in selectively damping the fluctuations of polar side groups.     

Sensing elements of optical sensors based on polystyrene with covalently immobilized reagents

A procedure was developed for the preparation of sensing elements for optical sensors based on pressed membranes of optically transparent polystyrene 250 μm in thickness. The procedure for assembling reagents on a matrix surface involved the operating sequence of nitration, reduction, diazotization, and azo coupling with organic reagents from the class of monoazochromotropic acid. This allowed us to prepare sensing elements with reproducible properties. The optical properties of immobilized reagents and their complexes with metal ions are practically analogous to those of compounds formed in solutions. External diffusion is a rate-determining step in the reactions with metal ions. The time constants of various sensing elements at metal concentrations of 4 × 10^?7 mol/mL were about 30 s, and the time taken to produce a signal at a 95% level was 50–56 s.