We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.
A. Biotinylation of DNA
Plasmid DNA were covalently biotinylated with guanine-specific biotin labels as described by the manufacturer (Mirus Bio, Madison, WI) but scaled to have ~1-2 biotin labels per DNA. Plasmid DNA (pEGFP-C1, 4.9 kb, Clontech, Mountain View, CA) was labeled in this protocol.
For 100μg DNA reaction:
DNase-free and RNase-free water | 75 μL |
10X Labeling Buffer A | 20 μL |
1μg/μL DNA | 100 μL |
LabelIT reagent | 5 μL |
Total Volume | 200 μL |
Note: Gel filtration based columns may lead to high UV absorbance or fluorescence background, which may affect the DNA quantification or fluorescence characterization.
Note: The level of biotinylation may be determined by HABA-based tests.
B. Labeling of the Cy5-Cationic Polymer
Chitosan (390 kDa, 83.5% deacetylated, Vanson, Redmond, WA) was used as a model cationic polymer in this study. The free primary amines on the chitosan polymer backbone were labeled with Cy5-NHS (Amersham Biosciences, Piscataway, NJ).
Note: In this study, a standard curve is constructed by measuring the emission intensity of Cy5-NHS ester at 670 nm. Characterize the labeling density by measuring the obtained emission at 670 nm from Cy5-labeled chitosan in the standard curve. Absorbance may also be used to determine the labeling efficiency but was not performed here.
C. Preparation of QD-labeled DNA and Cy5-Polymer
The molar ratio of pDNA to QD was kept in excess (pDNA : QD ≈ 1 : 2) to ensure complete conjugation of QDs to pDNA. The number of QDs labeled onto each pDNA can be estimated through TEM imaging or other equivalent facilities. In our study, the number of QDs per pDNA is estimated to be ~1-3 by TEM and single molecule spectroscopy.1 Use Millipore Milli-Q gradient water (>18.0 MW, 0.2um filtered) during the preparation.
Note: Keep the reaction in dark to prevent possible photobleaching.
Important: Be cautious to use the Qdot 605 ITK™ streptavidin conjugate (the ITK series), as Quantum dots in this catalog are designed for the purpose of FRET. The regular Qdot series are conjugated with a PEG layer to prevent non-specific binding, especially for cellular labeling. However, this additional coating enlarges the donor-acceptor distance, resulting in reduced energy transfer efficiency.
D. Fabrication of the SU-8 Masters Using Standard Photolithography
Important: Gradual ramping during the SU-8 master baking process is necessary, otherwise the SU-8 structure may detached from the silicon wafer or cracks on the SU-8 structure may be induced by stress-release.
E. Replica Molding of PDMS from the masters and Bonding to the Cover Glass
Important: Plasma treatment and overnight baking are essential to enhanced bonding strength.
F. Monitor the formation of DNA Nanocomplexes In the Microfluidic Device
Figure 1. QD-FRET provides a sensitive indication of the onset of DNA Nanocomplexes self-assembly
The authors have nothing to disclose.
Funding support provided by NIH grant HL89764, NSF grants 0546012, 0730503 and 0725528.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
SU-8 | MicroChem | SU-8 2025 | ||
PDMS | Dow Corning | Sylgard 184 | ||
Plasmid DNA | Clontech | pEGFP-C1 | 4.9 kb, MW = 3.3 x 106 | |
LabelIT Biotin Labeling Kit | Mirus Bio LLC | MIR 3400 | Standard protocol yields labeling efficiency of approximately one label every 20-60 bp of double-stranded DNA, The density of labeling was adjusted in this work. | |
Streptavidin 605QD | Invitrogen | Qdot® 605 ITK™ Streptavidin Conjugate | ||
Cy5-NHS Ester | Amersham Biosciences | PA15101 | ||
Chitosan | Vanson | 390 kDa | 83.5% deacetylated | |
Cover Glass | Fisher | 12-545C | No. 1; Size: 40 x 22mm | |
Gastight Glass Syringe | Hamilton | TLL series | 50μL to 500μL depending on sample volume | |
Tygon Tubing | SmallParts | 0.02 ID, 100ft | Tygon Tubes Microbore, 0.02 ID, 100ft | |
Connector | SmallParts | HTX-23R | Customized in length of 0.750″ | |
Syringe Pump | Harvard Apparatus | PHD-2000 | ||
CCD | Qimaging | Intensified Retiga Cooled | ||
Microscope | Olympus | BX-51 | 100W mercury arc lamp | |
ImageJ | NIH | v1.36b | http://rsb.info.nih.gov/ij | |
Origin Pro8 | OriginLab | Student Version | ||
Microscope Filter sets | Omega Optical | 475AF40 | Excitation filter in both channels | |
Microscope Filter sets | Omega Optical | 595AF60 | Emission filter in 605QD channel | |
Microscope Filter sets | Omega Optical | 670DF40 | Emission filter in QD-FRET channel | |
Microscope Filter sets | Omega Optical | 500 DRLP | Long pass dichroic in 605QD channel | |
Microscope Filter sets | Omega Optical | 595DRLP | Long pass dichroic in QD-FRET channel |