Mario Cabodi

Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications

Direct monitoring of primary molecular-binding interactions without the need for secondary reactants would markedly simplify and expand applications of high-throughput label-free detection methods. A simple interferometric technique is presented that monitors the optical phase difference resulting from accumulated biomolecular mass. As an example, 50 spots for each of four proteins consisting of BSA, human serum albumin, rabbit IgG, and protein G were dynamically monitored as they captured corresponding antibodies. Dynamic measurements were made at 26 pg/mm2 SD per spot and with a detectable concentration of 19 ng/ml. The presented method is particularly relevant for protein microarray analysis because it is label-free, simple, sensitive, and easily scales to high-throughput.

Evaporative Concentration on a Paper-Based Device to Concentrate Analytes in a Biological Fluid

We report the first demonstration of using heat on a paper device to rapidly concentrate a clinically relevant analyte of interest from a biological fluid. Our technology relies on the application of localized heat to a paper strip to evaporate off hundreds of microliters of liquid to concentrate the target analyte. This method can be used to enrich for a target analyte that is present at low concentrations within a biological fluid to enhance the sensitivity of downstream detection methods. We demonstrate our method by concentrating the tuberculosis-specific glycolipid, lipoarabinomannan (LAM), a promising urinary biomarker for the detection and diagnosis of tuberculosis. We show that the heat does not compromise the subsequent immunodetectability of LAM, and in 20 min, the tuberculosis biomarker was concentrated by nearly 20-fold in simulated urine. Our method requires only 500 mW of power, and sample flow is self-driven via capillary action. As such, our technology can be readily integrated into portable, battery-powered, instrument-free diagnostic devices intended for use in low-resource settings.

Quantification of surface etching by common buffers and implications on the accuracy of label-free biological assays

High throughput analyses in biochemical assays are gaining popularity in the post-genomic era. Multiple label-free detection methods are especially of interest, as they allow quantitative monitoring of biomolecular interactions. It is assumed that the sensor surface is stable to the surrounding medium while the biochemical processes are taking place. Using the Interferometric Reflectance Imaging Sensor (IRIS), we found that buffers commonly used in biochemical reactions can remove silicon dioxide, a material frequently used as the solid support in the microarray industry. Here, we report 53 pm to 731 pm etching of the surface silicon oxide over a 12-h period for several different buffers, including various concentrations of SSC, SSPE, PBS, TRIS, MES, sodium phosphate, and potassium phosphate buffers, and found that PBS and MES buffers are much more benign than the others. We observe a linear dependence of the etch depth over time, and we find the etch rate of silicon dioxide in different buffers that ranges from 2.73±0.76 pm/h in 1M NaCl to 43.54±2.95 pm/h in 6×SSC. The protective effects by chemical modifications of the surface are explored. We demonstrate unaccounted glass etching leading to erroneous results with label-free detection of DNA microarrays, and offer remedies to increase the accuracy of quantitative analysis.

Tunable Diacetylene Polymerized Shell Microbubbles as Ultrasound Contrast Agents

Monodisperse gas microbubbles, encapsulated with a shell of photopolymerizable diacetylene lipids and phospholipids, were produced by microfluidic flow focusing, for use as ultrasound contrast agents. The stability of the polymerized shell microbubbles against both aggregation and gas dissolution under physiological conditions was studied. Polyethylene glycol (PEG) 5000, which was attached to the diacetylene lipids, was predicted by molecular theory to provide more steric hindrance against aggregation than PEG 2000, and this was confirmed experimentally. The polymerized shell microbubbles were found to have higher shell-resistance than nonpolymerizable shell microbubbles and commercially available microbubbles (Vevo MicroMarker). The acoustic stability under 7.5 MHz ultrasound insonation was significantly greater than that for the two comparison microbubbles. The acoustic stability was tunable by varying the amount of diacetylene lipid. Thus, our polymerized shell microbubbles are a promising platform for ultrasound contrast agents.

Relationship between size and frequency dependent attenuation of monodisperse populations of lipid coated microbubbles

AbstractAcoustic techniques were employed to investigate the relationship between the mean size of monodispersions of lipid coated microbubbles and frequency dependent attenuation. Flow focusing microfluidic devices were constructed to produce several populations of monodisperse lipid coated microbubbles (i.e. with narrow size distribution). The mean diameter of each population was controlled by selecting the appropriate gas pressure and flowrate of the lipid solution through the device. Using this approach, monodispersions of lipid coated microbubbles were produced with mean diameters ranging from 3·3 to 8·3 μm. Using an acoustic spectroscopy technique, the frequency dependent attenuation coefficient for suspensions of lipid coated microbubbles was measured as a function of mean diameter. These results show that the width and peak magnitude of the attenuation spectrum depended upon the width of the microbubble size distribution (i.e. monodispersions versus polydispersions). Furthermore, as the mean size ...

Acoustic investigation of pressure-dependent resonance and shell elasticity of lipid-coated monodisperse microbubbles

In this study, frequency-dependent attenuation was measured acoustically for monodisperse lipid-coated microbubble suspensions as a function of excitation pressure and radius. The resonance frequency was identified from the attenuation spectra and had an inverse relationship with mean microbubble diameter and excitation pressure. A reduction in the estimated shell elasticity constant from 0.50 N/m to 0.29 N/m was observed as the excitation pressure was increased from 25 kPa to 100 kPa, respectively, which suggests a nonlinear relationship exists between lipid shell stiffness and applied strain. These findings support the viewpoint that lipid shells coating microbubbles exist as heterogeneous mixtures that undergo dynamic and rapid variations in mechanical properties under applied strains.

Monodisperse Micro-Oil Droplets Stabilized by Polymerizable Phospholipid Coatings as Potential Drug Carriers.

There is a critical need to formulate stable micron-sized oil droplets as hydrophobic drug carriers for efficient drug encapsulation, long-term storage, and sustained drug release. Microfluidic methods were developed to maximize the stability of micron-sized, oil-in-water (o/w) emulsions for potential use in drug delivery, using doxorubicin-loaded triacetin oil as a model hydrophobic drug formulation. Initial experiments examined multiple flow conditions for the dispersed (oil) and continuous (liposome aqueous) phases in a microfluidic device to establish the parameters that influenced droplet size. These data were fit to a mathematical model from the literature and indicate that the droplet sizes formed are controlled by the ratio of flow rates and the height of the device channel, rather than the orifice size. Next, we investigated effects of o/w emulsion production methods on the stability of the droplets. The stability of o/w emulsion produced by microfluidic flow-focusing techniques was found to be much greater (5 h vs 1 h) than for emulsions produced by mechanical agitation (vortexing). The increased droplet stability was attributed to the uniform size and lipid distribution of droplets generated by flow-focusing. In contrast, vortexed populations consisted of a wide size distribution that resulted in a higher prevalence of Ostwald ripening. Finally, the effects of shell polymerization on stability were investigated by comparing oil droplets encapsulated by a photopolymerizable diacetylene lipid shell to those with a nonpolymerizable lipid shell. Shell polymerization was found to significantly enhance stability against dissolution for flow-focused oil droplets but did not significantly affect the stability of vortexed droplets. Overall, results of these experiments show that flow-focusing is a promising technique for generating tunable, stable, monodisperse oil droplet emulsions, with potential applications for controlled delivery of hydrophobic drug formulations.

Sample concentration and purification for point-of-care diagnostics

The ability to increase the concentration of target analytes in a fixed sample volume can potentially lower the limit of detection for many biosensing techniques, and thus is key in sample preparation for infectious disease diagnosis. Concentration by evaporation is an effective method to achieve target enrichment. However, concentrating human samples, including blood and plasma, by evaporation-based methods is made challenging by high concentrations of proteins and electrolytes. Dehydration of the proteins causes the sample to turn into a gel, hindering further analysis. At the same time, decreasing the volume increases the overall concentration of electrolytes, causing bacterial or viral particle lysis, and making them more difficult to detect in affinity-based biosensors. Thus, we fabricated a microfluidic chip that incorporates both dialysis and concentration in a single design. The chip dialyzes the proteins from the plasma, while maintaining an appropriate concentration of electrolytes and concentrating the sample targets. The process to concentrate plasma or serum samples by a factor of 10 takes less than 30 minutes. As a proof-of-concept, we demonstrated the chip using a defective Human Immunodeficiency Virus (HIV). To distinguish patients on antiretroviral therapy who are failing therapy from those who are not, a diagnostic must be able to detect HIV in plasma down to at least 1000 particles per milliliter. For a number of technical reasons, it is difficult to get on-chip PCR reactions to reach this level of sensitivity, so concentration of HIV from lower viral load samples has the potential to improve the sensitivity of many types of molecular point-of-care viral load tests.

Pressure-dependent resonance frequency for lipid-coated microbubbles at low acoustic pressures

In this study, a flow-focusing microfluidic device was used to prepare lipid-coated microbubbles with a narrow size distribution. Size-controlled monodisperse microbubbles have been successfully produced with mean diameters ranging from 3.4–12.5 μm. These bubble populations were suspended in an exposure chamber and attenuation coefficients were measured by insonifing with 30-cycle narrow-band acoustic pulses at frequencies ranging from 0.5 to 4.5 MHz, and at four acoustic pressure amplitudes: 10 kPa, 20 kPa, 40 kPa, and 60 kPa. The resonance frequency was defined as the frequency where the peak attenuation coefficient was measured. It was shown that the resonance frequency decreased as the acoustic pressure increased, which was mainly due to the nonlinear behavior of the microbubbles. Compared with linear resonance frequency calculated for lipid-shelled microbubbles, the average changes of the resonance frequency at 10 kPa, 20 kPa, 40 kPa, and 60 kPa were 3.0%, −7.9%, −20.5%, and −27.6%, respectively.

National Institute of Biomedical Imaging and Bioengineering Point-of-Care Technology Research Network: Advancing Precision Medicine

To advance the development of point-of-care technology (POCT), the National Institute of Biomedical Imaging and Bioengineering established the POCT Research Network (POCTRN), comprised of Centers that emphasize multidisciplinary partnerships and close facilitation to move technologies from an early stage of development into clinical testing and patient use. This paper describes the POCTRN and the three currently funded Centers as examples of academic-based organizations that support collaborations across disciplines, institutions, and geographic regions to successfully drive innovative solutions from concept to patient care.To advance the development of point-of-care technology (POCT), the National Institute of Biomedical Imaging and Bioengineering established the POCT Research Network (POCTRN), comprised of Centers that emphasize multidisciplinary partnerships and close facilitation to move technologies from an early stage of development into clinical testing and patient use. This paper describes the POCTRN and the three currently funded Centers as examples of academic-based organizations that support collaborations across disciplines, institutions, and geographic regions to successfully drive innovative solutions from concept to patient care.