Evaluation of disposable microfluidic chip design for automated and fast Immunoassays.

We report here, the design and development of a disposable immunoassay chip for protein biomarker detection within ∼1 h. The unique design allows for real-time dynamic calibration of immunoassay for multiple biomarker detections on the chip. The limit of detection achieved for this test chip is 10 pg/ml for IL6, and 50 pg/ml for GFAP with a detection time of 1 h. The prototype instrument used for flowing the reagents through the chip can be easily assembled from off-the-shelf components with the final chemiluminescent detection carried out in a commercial plate reader. Optimization of different aspects of chip design, fabrication, and assay development is discussed in detail.

Development of Fully Automated Low-Cost Immunoassay System for Research Applications.

Enzyme-linked immunosorbent assay (ELISA) automation for routine operation in a small research environment would be very attractive. A portable fully automated low-cost immunoassay system was designed, developed, and evaluated with several protein analytes. It features disposable capillary columns as the reaction sites and uses real-time calibration for improved accuracy. It reduces the overall assay time to less than 75 min with the ability of easy adaptation of new testing targets. The running cost is extremely low due to the nature of automation, as well as reduced material requirements. Details about system configuration, components selection, disposable fabrication, system assembly, and operation are reported. The performance of the system was initially established with a rabbit immunoglobulin G (IgG) assay, and an example of assay adaptation with an interleukin 6 (IL6) assay is shown. This system is ideal for research use, but could work for broader testing applications with further optimization.

Multiplexed and fully automated detection of metabolic biomarkers using microdialysis probe.

We report here, the design and development of an automated near real-time continuous detection system for lactate, glutamate, pyruvate and glucose using microdialysis probe. The system developed can automatically push perfusate through microdialysis probe (20, 100 and 1000 kDa MWCO cutoff probe) at low to medium flow rate of 0.5-2 µL/min with almost 100% fluid recovery. The microdialysate collected from the probe is analyzed automatically for these four metabolite biomarkers. It operates in a continuous mode with measurements of all four biomarkers once every 20 min. The dynamic range for these different markers covers the entire clinical range of traumatic brain injury. The prototype shows a low variation of ~ 7-10% across the entire clinical range for all the biomarkers with fairly good accuracy of ~95%. The instrument canrun continuously for 24 h without user intervention. With a long tubing of 1 m to and from the microdialysis probe and associated dead volume, the total lag time for actual event at the probe site versusreported concentration is roughly 1 h.

A Low-Cost, Accurate, and High-Precision Fluid Dispensing System for Microscale Application.

We present here the development of a low-cost, accurate, and precise fluid dispensing system. It can be used with peristaltic or any other pump to improve the flow characteristics. The dispensing system has a range of 1 to 100 µL with accuracy of ~99.5% and standard deviation at ~150 nL over the entire range. The system developed does not depend on the accuracy or precision of the driving pump; therefore, any positive displacement pump can be used to get similar accuracy and precision, which gives an opportunity to reduce the cost of the system. The dispensing system does not require periodic calibration and can also be miniaturized for microfluidic application. Although primarily designed for aqueous liquid, it can be extended for different nonconductive liquids as well with modifications. The unit is further used for near real-time measurement of lactate from microdialysate. The individual components can easily be made disposable or sterilized for use in biomedical applications.

Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves.

Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.

Effects of separation length and voltage on isoelectric focusing in a plastic microfluidic device.

This paper describes the investigation on the effects of separation length and voltage on IEF in a plastic microfluidic device. A LIF, whole-channel imaging detection (WCID) system was developed to monitor proteins while they were moving under an electric field. IEF was carried out in a separation medium consisting of carrier ampholytes and a mixture of linear polymers (hydroxyethylcellulose and hydroxypropylcellulose). We found that the IEF separation resolution is essentially independent of separation length when the same voltage is applied, which agrees with the theory. This result supports the notion that IEF in a microfabricated device leads to more rapid analysis without sacrificing the resolving power. A higher separation voltage also brought about more rapid analysis and superior separation resolution. IEF of two proteins (green fluorescence protein and R-phycoerythrin) was achieved in 1.5 min when 500 V was applied across a 1.9-cm channel. We found that a linear relationship exists between the focusing time and the inverse of the electrical field strength. In addition, we confirmed the phenomenon in which the pH gradient was compressed to the middle of a channel, and we found that the relative amount of the gradient compression decreased with the channel length.

On the possibility of applying noncovalent dyes for protein labeling in isoelectric focusing.

Noncovalent fluorescent dyes are widely used for protein quantification and postcolumn detection in electrophoretic separations and recently some attempts to separate the precolumn labeled proteins using isoelectric focusing (IEF) have been made. In the present study, the possibility of applying the technique of protein labeling with noncovalent dyes for IEF is investigated. We found that fluorescent signal emitted by NanoOrange dye increases essentially in presence of carrier ampholyte (CA) components, which makes problematic a reliable protein detection in CA environment. Since in an isoelectric focusing mode the CA species are present in much greater concentration than the concentrations of fractionated proteins, the method of protein labeling with NanoOrange is not suitable for precolumn labeling and cannot be used for CA-IEF, at least without more detailed study of the dye-protein interaction mechanism.

Conductivity properties of carrier ampholyte pH gradients in isoelectric focusing.

The conductivity properties of natural pH gradient created by carrier ampholytes were studied during the process of isoelectric focusing (IEF). IEF was performed in capillaries (10-30 mm long) or in microchips with the same channel length. A 10-30x reduction of the conductivity of the separation medium was observed during the establishment of pH gradient. Results obtained using different IEF voltages indicate that there is a nonlinear relationship between the conductivity of an established pH gradient and the applied electric field. Our theoretical analysis using a simplified model generated values that reasonably agree with the experimental data. In addition, we found that above a certain electric field ( approximately 300 V/cm), resolution does not increase with the applied voltage as predicated; we observed band-broadening and gel breakdown. The approach presented in this work can be used for optimization of the IEF separation and judicious selection of IEF conditions.

Experimental and numerical analysis of short-pulse laser interaction with tissue phantoms containing inhomogeneities.

The objective is to perform an experimental and numerical study to analyze short-pulse laser propagation through tissue phantoms without and with inhomogeneities embedded in them. For a short-pulse laser the observed optical signal has a distinct temporal shape, and the shape is a function of the medium properties. The scattered temporal transmitted and reflected optical signals are measured experimentally with a streak camera for tissue phantoms irradiated with a short-pulse laser source. A parametric study involving different scattering and absorption coefficients of tissue phantoms and inhomogeneities, as well as the detector positions and orientations, is performed. The temporal and spatial profiles of the scattered optical signals are compared with the numerical modeling results obtained by solving the transient radiative transport equation by using the discrete ordinates technique.