Mohammad Jabasini

Trafficking and Subcellular Localization of Multiwalled Carbon Nanotubes in Plant Cells

Major barriers to delivery of biomolecules are crossing the cellular membranes and achieving a high cytoplasmic concentration by circumventing entrapment into endosomes and other lytic organelles. Motivated by such aim, we have investigated the capability of multiwalled carbon nanotubes (MWCNTs) to penetrate the cell membrane of plant protoplasts (plant cells made devoid of their cell walls via enzymatic treatment) and studied their internalization mechanism via confocal imaging and TEM techniques. Our results indentified an endosome-escaping uptake mode of MWCNTs by plant protoplasts. Moreover, short MWCNTs (<100 nm) were observed to target specific cellular substructures including the nucleus, plastids, and vacuoles. These findings are expected to have a significant impact on plant cell biology and transformation technologies.

DNA separation by microchip electrophoresis using low-viscosity hydroxypropylmethylcellulose-50 solutions enhanced by polyhydroxy compounds

Low‐viscosity polymer solutions have potential for double‐stranded (ds) DNA separations in micrototal analysis systems (ν‐TAS). In this paper, we report dilute, low‐viscosity hydroxypropylmethylcellulose‐50 (HPMC‐50, 11.5 kDa) solutions containing polyhydroxy additives as separation media. Predominant operational variables, such as applied electric field strength, fluorescent intercalator (YOPro‐1) concentration, polymer concentration, and additive concentration, are thoroughly investigated. Fast (within 170 s) and excellent separation of DNA restriction fragments ranging in size from 72 to 1353 base pairs (bp) is achieved in a 30 mm length channel of polymethylmethacrylate (PMMA) microchips at an electric field strength of 300 V/cm, by introducing 8% mannitol, 8% glucose or 10% glycerol additives into a 2% HPMC‐50 / 1×Tris‐borate‐EDTA (TBE) solution. The low‐viscosity (40 cP) matrix formulation provides both coating of the microchannels and separation of DNA in one step. The performance in the solution surpasses that in highly concentrated HPMC‐50 solution. In addition, separation using 1×Tris‐EDTA buffer in the 2% HPMC‐50 matrix containing polyhydroxy additives also exhibits a notably increased performance. This is presumably due to formation of hydrogen‐bonding interactions of polyhydroxy additives with HPMC‐50 matrix and DNA so as to increase the coupling interactions between matrix and DNA molecules during electrophoresis. The result reflects that boric acid is not a prerequisite in polyhydroxy‐enhanced HPMC‐50 solution for separation.

Analysis of DNA polymorphisms on the human Y-chromosome by microchip electrophoresis.

Validation of microchip electrophoresis in DNA analysis has been carried out using an Agilent 2100 Bioanalyzer. With a DNA 500 Assay Kit, the reproducibility and accuracy of fragment sizing of a 10 bp DNA ladder have been shown to be satisfactory with the relative standard deviation and the relative error mostly below 1.0 and 5.0% (n = 12), respectively. Both intraday and interday validations of fragment sizing and quantitation have also been performed with a 7500 Assay Kit (n = 48). Although the results of quantitation are not as good as that of sizing, due to the manual introduction of samples and markers into the chip wells, they are still sufficient to carry out further analyses of practical samples. Based on such reliable results, fast analysis of DNA polymorphisms on the human Y‐chromosome has been realized with microchip electrophoresis. The total analysis times of three genomic polymorphisms on the Y‐chromosome, Y Alu polymorphism, 47z/StuI, and 12f2, are all within 100 s, and the relative standard deviation and relative error of fragment sizes are below 3.5 and 3.7%, respectively. In addition, a mixture of nine DNA markers on the human Y‐chromosome related to examine the cause of spermatogenic failure have been separated successfully with the smallest fragment size difference of 7 bp. Our results demonstrate the potential of microchip electrophoresis in polymorphism analysis with the advantages of high speed, good reproducibility, high precision, and high resolution.

Reduced viscosity polymer matrices for microchip electrophoresis of double-stranded DNA

On a polymethylmethacrylate (PMMA) microchip, double-stranded DNA fragments with a wide size range from 50 bp to 20 kbp were separated by two polymer solutions. One was a hydroxypropylmethylcellulose-4000 (HPMC-4000) solution of 1.3% (w/v) to separate fragments below 590 bp, and another was a mixed four molecular weight poly(ethylene oxide) solution at a total concentration of 0.1% to separate fragments above 520 bp. The widths at half height (wh) of the fragments had a good relationship with their migration times (tR) in both polymer solutions. Such a relationship was suitable for obtaining the wh values of unresolved peaks, calculating the resolution of two adjacent fragments, and optimizing microchip separation matrices. Based on the relativity, a low viscosity medium containing 2% HPMC-50 and 8% glucose was optimized for high-performance separation of a phiX174 HaeIII restriction fragment digest.

Microchip analysis of plant glucosinolates

We describe a new and selective analytical method for the separation and quantitation of plant glucosinolates. The new method, which utilizes microchip CE (μ‐CE) with fluorescence detection, circumvents the multistep procedures characteristic of conventional methods. Glucosinolates form charge transfer complexes with the xanthene dyes phloxine‐B and eosin‐B. The glucosinolates–phloxine‐B complex cannot be excited at 470 nm. Thus, the decrease in peak intensity of phloxine‐B after complex formation is used to quantitatively measure total glucosinolates in Arabidopsis thaliana seeds. For qualitative analysis, complex formation with eosin‐B is used. The sensitivity of eosin‐B detection at excitation/emission 470 nm/540 nm was low. However, sensitivity increased following complex formation with sinigrin (≥3 μg/mL). A batch‐learning, self‐organizing map was applied to visualize and organize analytical data into 2‐D matrix with similar and related data clustered together or near each other. This organized matrix was used to optimize electrophoretic conditions for the analysis. This study suggests potential applications of μ‐CE in plant metabolomics analyses without use of labeling fluorophores.

Anomalous Separation of Small Y-Chromosomal DNA Fragments on Microchip Electrophoresis

We investigated an anomalous DNA separation where two DNA fragments from the human Y-chromosome sY638 (64 bp) and sY592 (65 bp), with only one base pair difference, were separated. This result is abnormal since in a previous study, we found that 5 bp was the minimum difference between two DNA fragments that the microchip electrophoresis system can separate. The formation of a mini-loop in the structure of the DNA fragment of sY638 (64 bp) was strongly expected to be the reason. To investigate this, we synthesized three modified DNA fragments for sY638 (64 bp), and the modifications were in two expected locations for possible mini-loop formation. Later, the separation between sY592 (65 bp) and the three modified fragments of sY638 (64 bp) was not possible. Thus, we conclude that the formation of a mini-loop in the structure of the DNA is the reason behind this anomalous separation.

Microfabricated Chip Electrophoresis Technology for DNA Analysis

In this report, two microfabricated chip electrophoresis techniques and several application studies were tested for rapid and high-resolution separation of double-stranded (ds)DNA. In one technique, low-viscosity hydroxypropylmethylcellulose-50 (HPMC-50) matrix accompanied by polyhydroxy compounds, such as mannitol, glucose, and glycerol, showed higher resolving power than conventionally and singly used HPMC-50 matrix. The new matrix is easy to be hyphenated in the future μ-TAS platforms. Another technique is through the stepwise (multi-step) filling of different concentrations of one polymer or different types of polymers to achieve high-resolution separation of both short and long DNA fragments simultaneously. The technique has good migration-time reproducibility for the analysis of restriction digest fragments and ladders. The separation application of some PCR products was performed on an Agilent 2100 Bioanalyzer. The reproducibility and accuracy of fragment sizing of a DNA ladder were satisfactory. Fast analysis of DNA polymorphisms on the human Y-chromosome was realized with the analytical time of three genomic polymorphisms on the Y-chromosome (Y Alu polymorphism, 47z/StuI and 12 f2) to be within 110 s, respectively. A mixture of nine DNA markers on the human Y-chromosome related to examine the cause of spermatogenic failure was successfully separated with the smallest fragment size difference of 7 bp.