Performance of an integrated microoptical system for fluorescence detection in microfluidic systems

This article presents a new integrated microfluidic/microoptic device designed for basic biochemical analysis. The microfluidic network is wet-etched in a Borofloat 33 (Pyrex) glass wafer and sealed by means of a second wafer. Unlike other similar microfluidic systems, elements of the detection system are realized with the help of microfabrication techniques and directly deposited on both sides of the microchemical chip. The detection system is composed of the combination of refractive circular or elliptical microlens arrays and chromium aperture arrays. The microfluidic channels are 60 microm wide and 25 microm deep. The elliptical microlenses have a major axis of 400 microm and a minor axis of 350 microm. The circular microlens diameters range from 280 microm to 350 microm. The apertures deposited on the outer chip surfaces are etched in a 3000-A-thick chromium layer. The overall thickness of this microchemical system is < 1.6 mm. A limit of detection of 3.3 nM for a Cy5 solution in phosphate buffer (pH 7.4) was demonstrated. The cross-talk signal measured between two adjacent microchannels with 1 mm pitch was < 1:5600, meaning that < or = 1.8 x 10(-4)% of the fluorescence light power emitted from one microchannel filled with a 50 microM Cy5 solution reaches the photodetector at the adjacent microchannel. This performance compares very well with that obtainable in microchemical chips using confocal fluorescence systems, taking differences in parameters, such as excitation power into microchannels, data acquisition rates, and signal filtering into account.     

Analysis of lipoproteins by capillary zone electrophoresis in microfluidic devices: assay development and surface roughness measurements

The development of a new assay for lipoproteins by capillary electrophoresis in fused-silica capillaries and in glass microdevices is described in this paper. The separation of low-density (LDL) and high-density (HDL) lipoproteins by capillary zone electrophoresis is demonstrated in fused-silica capillaries with both UV absorption and laser-induced fluorescence detection. This separation was accomplished using Tricine buffer (pH 9.0) with methylglucamine added as a dynamic coating. With UV detection, LDL eluted as a relatively sharp peak with a migration time of approximately 11 min and HDL eluted as a broad peak with a migration time of 12.5 min. Fluorescence detection of lipoproteins stained with NBD-ceramide was used with the same buffer system to give comparable results. Furthermore, fluorescence staining of human serum samples yielded results similar to the fluorescently stained LDL and HDL fractions, showing that this method can be used to quantify lipoproteins in serum samples. The method was also used to detect lipoproteins in glass micro-CE devices. Very similar results were obtained in microdevices although with much faster analysis times, LDL eluted as a sharp peak at approximately 25 s and HDL as a broad peak at slightly longer time. In addition, higher resolution was obtained on chips. To our knowledge, these results show the first separation and detection of lipoproteins in a microfluidic device using native serum samples. Atomic force microscopy was used to characterize the rms surface roughness (Rq) of microfluidic channels directly. Devices with different surface roughness values were fabricated using two different etchants for Pyrex wafers with a polysilicon masking layer. Using 49% HF, the measured roughness is Rq = 10.9 +/- 1.6 nm and with buffered HF (NH4F + HF) the roughness is Rq = 2.4 +/- 0.7 nm. At this level of surface roughness, there is no observable effect on the performance of the devices for this lipoprotein separation.     

Autonomous microfluidic capillary system

The transport of minute amounts of liquids using microfluidic systems has opened avenues for higher throughput and parallelization of miniaturized bio/chemical processes combined with a great economy of reagents. In this report, we present a microfluidic capillary system (CS) that autonomously transports aliquots of different liquids in sequence: liquids pipetted into the service port of the CS flow unidirectionally through the various sections of the CS, which comprises a 15-pL reaction chamber, into the capillary pump. A CS can thus be operated by simply delivering the different samples to its service port. The liquid transport concept presented here is advantageous because the pumping and valving functions are integrated into the device by means of capillary phenomena, and it therefore does not require any external power supply or control device. Thus, arrays of CSs can easily be formed by cloning a functional CS. Alternatively, the flow of liquids in CSs can also be interactively tuned if desired by (i) forcing the evaporating of liquid out of the capillary pumps and (ii) by contacting a secondary, removable capillary pump to the embedded ones. We illustrate the possibilities of CSs by conducting a surface immunoassay for a cardiac marker, within 25 min, on an area of 100 x 100 microm2, using 16 sequential filling steps.     

Optical observations of acoustical radiation force effects on individual air bubbles

Previous studies dealing with contrast agent microbubbles have demonstrated that ultrasound (US) can significantly influence the movement of microbubbles. In this paper, we investigated the influence of the acoustic radiation force on individual air bubbles using high-speed photography. We emphasize the effects of the US parameters (pulse length, acoustic pressure) on different bubble patterns and their consequences on the translational motion of the bubbles. A stream of uniform air bubbles with diameter ranging from 35 microm to 79 microm was generated and insonified with a single US pulse emitted at a frequency of 130 kHz. The bubble sizes have been chosen to be above, below, and at resonance. The peak acoustic pressures used in these experiments ranged from 40 kPa to 120 kPa. The axial displacements of the bubbles produced by the action of the US pulse were optically recorded using a high-speed camera at 1 kHz frame rate. The experimental results were compared to a simplified force balance theoretical model, including the action of the primary radiation force and the fluid drag force. Although the model is quite simple and does not take into account phenomena like bubble shape oscillations and added mass, the experimental findings agree with the predictions. The measured axial displacement increases quasilinearly with the burst length and the transmitted acoustic pressure. The axial displacement varies with the size and the density of the air bubbles, reaching a maximum at the resonance size of 48 microm. The predicted displacement values differ by 15% from the measured data, except for resonant bubbles for which the displacement was overestimated by about 40%. This study demonstrates that even a single US pulse produces radiation forces that are strong enough to affect the bubble position.     

The radiation mode theory in ultrasonics

This paper describes the history and the state of the art in radiation mode theory (RMT) in ultrasonics. The RMT originates from electromagnetism in which it has proved to be very efficient in the field of wave guides and discontinuities. In ultrasonics, the RMT made its entrance only a decade ago and has already proved to be very efficient in describing the interaction of sound with discontinuities such as a step on a plate, a liquid wedge, the extremity of a plate and much more. It is likely that the development of the RMT for two-dimensional (2-D) isotropic media has come almost to an end. This paper lists the results obtained so far. Further extensions to more complicated media are to be expected in the coming decade.     

Ultrasound-induced gas release from contrast agent microbubbles

We investigated gas release from two hardshelled ultrasound contrast agents by subjecting them to high-mechanical index (MI) ultrasound and simultaneously capturing high-speed photographs. At an insonifying frequency of 1.7 MHz, a larger percentage of contrast bubbles is seen to crack than at 0.5 MHz. Most of the released gas bubbles have equilibrium diameters between 1.25 and 1.75 microm. Their disappearance was observed optically. Free gas bubbles have equilibrium diameters smaller than the bubbles from which they have been released. Coalescence may account for the long dissolution times acoustically observed and published in previous studies. After sonic cracking, the cracked bubbles stay acoustically active.     

Protein flexibility and ligand rigidity: a thermodynamic and kinetic study of ITAM-based ligand binding to Syk tandem SH2

The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI-MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the gamma chain of the FcepsilonRI-receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 degrees C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of approximately 9 kcal mol-1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI-MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two-step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal-transduction processes.     

Multifrequency transducer for microemboli classification and sizing

The classification of circulating microemboli as gaseous or particulate matter is essential to establish the relevance of the detected embolic signals. Until now, Doppler techniques have failed to determine unambiguously the nature of circulating microemboli. Recently, a new approach based on the analysis of radio frequency (RF) signal and using the nonlinear characteristics of gaseous bubbles to classify emboli was investigated. The main limitation of these studies was the requirement of two separate transducers for transmission and reception. This paper presents a multi-frequency transducer with two independent transmitting elements and a separate receiving part with a wide frequency band. The transmitting elements are positioned in a concentric design and cover a frequency band between 100 and 600 kHz. The receiving part consists of a polyvinylidene fluoride layer. The new transducer has been tested in vitro using gaseous emboli. It could correctly classify and size air emboli with diameters ranging from 10 microm to 105 microm.     

Noise equalization for detection of microcalcification clusters in direct digital mammogram images

Equalizing image noise is shown to be an important step in the automatic detection of microcalcifications in digital mammography. This study extends a well established film-screen noise equalization scheme developed by Veldkamp et al. for application to full-field digital mammogram (FFDM) images. A simple noise model is determined based on the assumption that quantum noise is dominant in direct digital X-ray imaging. Estimation of the noise as a function of the gray level is improved by calculating the noise statistics using a truncated distribution method. Experimental support for the quantum noise assumption is presented for a set of step wedge phantom images. Performance of the noise equalization technique is also tested as a preprocessing stage to a microcalcification detection scheme. It is shown that the square root model based approach which FFDM allows leads to a robust estimation of the high frequency image noise. This provides better microcalcification detection performance when compared to the film-screen noise equalization method developed by Veldkamp. Substantially better results are obtained than when noise equalization is omitted. A database of 124 direct digital mammogram images containing 28 microcalcification clusters was used for evaluation of the method.     

Surface heat transfer coefficients to stationary spherical particles in an experimental unit for hydrofluidisation freezing of individual foods

Convection heat transfer to spherical particles inside a hydrofluidisation freezing unit was investigated. The unit contained a food tank with a perforated bottom plate to create agitating jets. An aqueous solution of 30% ethanol+20% glucose was used as the refrigeration medium in a temperature range of −20 to 0 °C and flow rates from 5 to 15 l min⁻¹. The lumped capacitance method was applied on cooling profiles of aluminium spheres of 5–50 mm to obtain surface heat transfer coefficients. Coefficients were within a range of 154–1548 W m⁻² °C⁻¹, and depended on diameter, flow rate, refrigeration temperature and fluid agitation level. The agitation due to jets was accounted for by means of an agitation Reynolds number in a Nusselt correlation A large variability of measured surface heat transfer coefficients was observed. This could be attributed to non-constant flow and turbulence fields in the refrigeration medium. The value of the heat transfer coefficient was compared to values determined on strawberries.