Po-Jung Huang

Use of a micro- to nanochannel for the characterization of surface-enhanced Raman spectroscopy signals from unique functionalized nanoparticles

Abstract. A micro- to nanochannel nanoparticle aggregating device that does not require any input energy to organize the particles to a specific location, i.e., no pumps, plugs, heat, or magnets, has been designed and used to characterize the surface-enhanced Raman spectroscopy (SERS) signal from four unique functionalized nanoparticles (gold, silver-gold nanocages, silver nanocubes, and silica-gold nanoshells). The SERS signal was assessed in terms of the peak signal strength from the four different Raman reporter functionalized nanoparticles to determine which nanoparticle had better utility in the channel to provide the most robust platform for a future biological analyte detection device. The innovation used to fabricate the micro- to nanochannel device is described; the TEM images of the nanoparticles are shown; the absorption data for the nanoparticles are given; and the spectral data for the Raman reporter, mercaptobenzoic acid (MBA), are depicted. In the micro- to nanochannel described in this work, 5  μl of 22.3  μM MBA functionalized silver nanocubes were determined to have the strongest SERS enhancement.

Comparison of Fe2O3 and Fe2CoO4 core-shell plasmonic nanoparticles for aptamer mediated SERS assays

Conjugation of oligonucleotides or aptamers and their corresponding analytes onto plasmonic nanoparticles mediates the formation of nanoparticle assemblies: molecularly bound bundles of nanoparticles which cause a measurable change in the colloid’s optical properties. Here, we present further optimization of a “SERS off” competitive binding assay utilizing plasmonic and magnetic nanoparticles for the detection of the toxin bisphenol A (BPA). The assay involves 1) a ‘target’ silver nanoparticle functionalized with a Raman reporter dye and PEGylated BPA-binding DNA aptamers, and 2) a version of the toxin BPA, bisphenol A diglycidyl ether (BADGE), PEGylated and immobilized onto a silver coated magnetic ’probe’ nanoparticle. When mixed, these target and probe nanoparticles cluster into magnetic dimers and trimers and an enhancement in their SERS spectra is observed. Upon introduction of free BPA in its native form, target AgNPs are competitively freed; reversing the nanoparticle assembly and causing the SERS signal to “turn-off” and decrease in response to the competitive binding event. The assay particles were housed inside two types of optofluidic chips containing magnetically active nickel pads, in either a straight or spotted pattern, and both Fe2O3 and Fe2CoO4 were compared as magnetic cores for the silver coated probe nanoparticle. We found that the Ag@ Fe2O3 particles were, on average, more uniform in size and more stable than Ag@ Fe2CoO4, while the addition of cobalt significantly improved the collection time of particles within the magnetic chips. Using 3D Raman mapping, we found that the straight channel design with the Ag@ Fe2O3 particles provided the most uniform nanoparticle organization, while the spotted channel design with Ag@ Fe2CoO4 demonstrated a larger SERS enhancement, and thus a lower limit of detection.

Fabrication of Bacteria Environment Cubes with Dry Lift-Off Fabrication Process for Enhanced Nitrification.

We have developed a 3D dry lift-off process to localize multiple types of nitrifying bacteria in polyethylene glycol diacrylate (PEGDA) cubes for enhanced nitrification, a two-step biological process that converts ammonium to nitrite and then to nitrate. Ammonia-oxidizing bacteria (AOB) is responsible for converting ammonia into nitrite, and nitrite-oxidizing bacteria (NOB) is responsible for converting nitrite to nitrate. Successful nitrification is often challenging to accomplish, in part because AOB and NOB are slow growers and highly susceptible to many organic and inorganic chemicals in wastewater. Most importantly, the transportation of chemicals among scattered bacteria is extremely inefficient and can be problematic. For example, nitrite, produced from ammonia oxidation, is toxic to AOB and can lead to the failure of nitrification. To address these challenges, we closely localize AOB and NOB in PEGDA cubes as microenvironment modules to promote synergetic interactions. The AOB is first localized in the vicinity of the surface of the PEGDA cubes that enable AOB to efficiently uptake ammonia from a liquid medium and convert it into nitrite. The produced nitrite is then efficiently transported to the NOB localized at the center of the PEGDA particle and converted into non-toxic nitrate. Additionally, the nanoscale PEGDA fibrous structures offer a protective environment for these strains, defending them from sudden toxic chemical shocks and immobilize in cubes. This engineered microenvironment cube significantly enhances nitrification and improves the overall ammonia removal rate per single AOB cell. This approach—encapsulation of multiple strains at close range in cube in order to control their interactions—not only offers a new strategy for enhancing nitrification, but also can be adapted to improve the production of fermentation products and biofuel, because microbial processes require synergetic reactions among multiple species.

Pneumatically Actuated Soft Micromold Device for Fabricating Collagen and Matrigel Microparticles

Collagen microparticles have recently gained more attention as viable cell confinement blocks in many biomedical research fields. Small volume and high surface area of collagen structure improve cell confinement, viability, and proliferation. Moreover, dense collagen fiber structure can protect cells from immune destruction. The ability to produce collagen microparticles in an accurate and reliable way is of upmost importance to the advancement of many biomedical researches, especially cancer research and tissue engineering. Currently, no such fabrication technique exists due to inherent fragility of collagen. Herein, we report the very first platform, pneumatically actuated soft micromold (PASMO) device, which addresses challenges in collagen microparticle production. Our new platform uses a soft micromold with a pneumatic actuator that can produce arbitrary shapes of collagen microstructures precisely from 100 μm to over 2 mm in range and can encapsulate cells inside without damaging the shape. The duplication accuracy of more than 96% in dimensions and 90% in depth has been demonstrated. The density of collagen fiber distribution is determined to be 86.57%, which is higher than that of collagen microparticles produced by other methods. We have confirmed cell viability in collagen microparticles. We also produce Matrigel™ particles as tool to develop a xenograft cancer model. The results demonstrate that Matrigel particles created by the PASMO device can reduce cell scattering for the xenograft model and the uniformity of tumors developed in mice is 12-fold improved, which can lead to an increased accuracy of cancer metastasis studies and drug screening research. These breakthroughs in the production of modular microparticles will push the boundaries of cancer research in the near future.

A magneto-fluidic nanoparticle trapping platform for surface-enhanced Raman spectroscopy

A microfluidic device utilizing magnetically activated nickel (Ni) micropads has been developed for controlled localization of plasmonic core-shell magnetic nanoparticles, specifically for surface enhanced Raman spectroscopy (SERS) applications. Magnetic microfluidics allows for automated washing steps, provides a means for easy reagent packaging, allows for chip reusability, and can even be used to facilitate on-chip mixing and filtration towards full automation of biological sample processing and analysis. Milliliter volumes of gold-coated 175-nm silica encapsulated iron oxide nanoparticles were pumped into a microchannel and allowed to magnetically concentrate down into 7.5 nl volumes over nano-thick lithographically defined Ni micropads. This controlled aggregation of core-shell magnetic nanoparticles by an externally applied magnetic field not only enhances the SERS detection limit within the newly defined nanowells but also generates a more uniform (∼92%) distribution of the SERS signal when compared to random mechanical aggregation. The microfluidic flow rate and the direction and strength of the magnetic field determined the overall capture efficiency of the magneto-fluidic nanoparticle trapping platform. It was found that a 5 μl/min flow rate using an attractive magnetic field provided by 1 × 2 cm neodymium permanent magnets could capture over 90% of the magnetic core-shell nanoparticles across five Ni micropads. It was also observed that the intensity of the SERS signal for this setup was 10-fold higher than any other flow rate and magnetic field configurations tested. The magnetic concentration of the ferric core-shell nanoparticles causes the SERS signal to reach the steady state within 30 min can be reversed by simply removing the chip from the magnet housing and sonicating the retained particles from the outlet channel. Additionally, each magneto-fluidic can be reused without noticeable damage to the micropads up to three times.

Novel 3D coaxial flow-focusing nozzle device for the production of monodispersed collagen microspheres

We have developed a 3D coaxial flow-focusing nozzle device for the mass production of monodispersed collagen microspheres and chemically crosslinked them using EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) and N-hydroxysuccinimide (NHS). The size of the microspheres was varied between 200 μm and 600 μm by adjusting the ratio of the flow rates of the dispersed and continuous phases. MDA231-GFP cells were attached to the surface of these particles and their viability was investigated. Because they are comprised of a natural biomaterial, these collagen microspheres will have numerous applications, including bone regeneration scaffolds for tissue engineering and analyses of cancer cell interactions in a 3D environment.

Development of automated high throughput single molecular microfluidic detection platform for signal transduction analysis

Signal transductions including multiple protein post-translational modifications (PTM), protein-protein interactions (PPI), and protein-nucleic acid interaction (PNI) play critical roles for cell proliferation and differentiation that are directly related to the cancer biology. Traditional methods, like mass spectrometry, immunoprecipitation, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy require a large amount of sample and long processing time. “microchannel for multiple-parameter analysis of proteins in single-complex (mMAPS)”we proposed can reduce the process time and sample volume because this system is composed by microfluidic channels, fluorescence microscopy, and computerized data analysis. In this paper, we will present an automated mMAPS including integrated microfluidic device, automated stage and electrical relay for high-throughput clinical screening. Based on this result, we estimated that this automated detection system will be able to screen approximately 150 patient samples in a 24-hour period, providing a practical application to analyze tissue samples in a clinical setting.

Development of an optofluidic SERS-based biomedical sensor

Rapid assessment of radiation exposure to sensitive organs like the gut is extremely important for large populations exposed to ionized radiation, for instance during warfare. Recent results have shown that plasma citrulline levels appear to track gut function after irradiation levels in mice and humans. The current ways to monitor blood citrulline levels are bulky, laborious, time-consuming and expensive methods. Therefore, an optofludic point-of-care (POC) system using surface enhanced Raman spectroscopy to measure plasma citrulline as a marker for radiation exposure that overcomes the above issues is being developed. As a first step toward development of this system four colloidal nanoparticles, spherical gold, silver cubes, silica-gold nanoshells, and silver-gold nanocages have been analyzed for use in the POC system. Transmission electron microscopy (TEM) images have been taken of each nanoparticle to visualize the morphology of the nanoparticles, which is vital for SERS. Ultraviolet-visible (UV/Vis) spectroscopy was also collected to verify the extinction spectra for each nanoparticle was in resonance with the excitation wavelength. The nanoparticles were functionalized with mercaptobenzoic acid (MBA), a Raman reporter molecule, and SERS spectra were collected to determine which has better utility in a novel micro-to-nanochannel. The data showed that the silver nanocubes have a larger enhancement factor than the gold nanospheres, nanoshells, or nanocages. Currently, these nanocubes are being functionalized with the citulline for assessing the concentration sensitivity and dynamic range for ultimate use as a marker for radiation.

Nanofluidic Strategies for Cancer Research

Nanofluidic system can be used as powerful tool for detecting single molecules through fluorescence correlation spectroscopy (FCS). Several types of nanofluidic channels, such as hollow nanofibres or nanotrenches, can be constructed on quartz wafers, though electrospinning and nanolithography, respectively. The advantages of nanofluidic channels in molecule detection are not only in reducing the amount of volume of analyte, but also for improving electrokinetic molecule transport. Therefore, small molecules, like proteins or DNA, can be detected in nanochannels. Furthermore, nanofluidic channels can be used to monitor protein–protein, post translational modification, protein–DNA, and protein–RNA interactions, which rely on labelling proteins of interest within fluorescent molecules, in tissue samples directly. Based on these results, nanofluidic channels can be used in diagnostic application for early diagnosis of cancers and drug screenings.

Ferric plasmonic nanoparticles, aptamers, and magnetofluidic chips: toward the development of diagnostic surface-enhanced Raman spectroscopy assays

Abstract. Conjugation of aptamers and their corresponding analytes onto plasmonic nanoparticles mediates the formation of nanoparticle assemblies: molecularly bound nanoclusters that cause a measurable change in the colloid’s optical properties. The optimization of a surface-enhanced Raman spectroscopy (SERS) competitive binding assay utilizing plasmonic “target” and magnetic “probe” nanoparticles for the detection of the toxin bisphenol-A (BPA) is presented. These assay nanoclusters were housed inside three types of optofluidic chips patterned with magnetically activated nickel pads, in either a straight or array pattern. Both Fe2O3 and Fe2CoO4 were compared as potential magnetic cores for the silver-coated probe nanoparticles. We found that the Ag@Fe2O3 particles were, on average, more uniform in size and more stable than Ag@Fe2CoO4, whereas the addition of cobalt significantly improved the collection time of particles. Using Raman mapping of the assay housed within the magnetofluidic chips, it was determined that a 1×5 array of 50  μm square nickel pads provided the most uniform SERS enhancement of the assay (coefficient of variation ∼25%) within the magnetofluidic chip. Additionally, the packaged assay demonstrated the desired response to BPA, verifying the technology’s potential to translate magnetic nanoparticle assays into a user-free optical analysis platform.