Probing and quantifying DNA-protein interactions with asymmetrical flow field-flow fractionation.

Tools capable of measuring binding affinities as well as amenable to downstream sequencing analysis are needed for study of DNA-protein interaction, particularly in discovery of new DNA sequences with affinity to diverse targets. Asymmetrical flow field-flow fractionation (AF4) is an open-channel separation technique that eliminates interference from column packing to the non-covalently bound complex and could potentially be applied for study of macromolecular interaction. The recovery and elution behaviors of the poly(dA)n strand and aptamers in AF4 were investigated. Good recovery of ssDNAs was achieved by judicious selection of the channel membrane with consideration of the membrane pore diameter and the radius of gyration (Rg) of the ssDNA, which was obtained with the aid of a Molecular Dynamics tool. The Rg values were also used to assess the folding situation of aptamers based on their migration times in AF4. The interactions between two ssDNA aptamers and their respective protein components were investigated. Using AF4, near-baseline resolution between the free and protein-bound aptamer fractions could be obtained. With this information, dissociation constants of ∼16nM and ∼57nM were obtained for an IgE aptamer and a streptavidin aptamer, respectively. In addition, free and protein-bound IgE aptamer was extracted from the AF4 eluate and amplified, illustrating the potential of AF4 in screening ssDNAs with high affinity to targets. Our results demonstrate that AF4 is an effective tool holding several advantages over the existing techniques and should be useful for study of diverse macromolecular interaction systems.

Dissociation-based screening of nanoparticle-protein interaction via flow field-flow fractionation.

A protein corona will be formed on nanoparticles (NPs) entering a biological matrix, which can influence particles' subsequent behaviors inside the biological systems. For proteins bound stably to the NPs, they can exhibit different association/dissociation rates. The binding kinetics could affect interaction of the NPs with cell surface receptors and possibly contribute to the outcomes of NPs uptake. In the present study, a method to differentiate the corona proteins based on their relative dissociation rates from the NPs was developed, employing flow field-flow fraction (F4) in combination with centrifugation. The proteins bound to the superparamagnetic iron oxide NPs (SPION) present in an IgG/albumin depleted serum were isolated via collection of the SPIONs by either F4 or centrifugation. They were subsequently analyzed by LC-MS/MS and identified. Because the SPION-protein complexes injected to F4 dissociated continuously under the nonequilibrium separation condition, only the proteins with slow enough dissociation rates would be collected with the NPs in the eluent of F4. However, in centrifugation, proteins with good affinity to the SPIONs were collected regardless of the dissociation rates of the complexes. In both cases, the nonbinding ones were washed off. Capillary electrophoresis and circular dichroism were employed to verify the binding situations of a few SPION-protein interactions, confirming the effectiveness of our method. Our results support that our method can screen for proteins binding to NPs with fast on-and-off rates, which should be the ones quickly exchanging with the free matrix proteins when the NPs are exposed to a new biological media. Thus, our method will be useful for investigation of the temporal profile of protein corona and its evolution in biological matrices as well as for high-throughput analysis of the dynamic feature of protein corona related to particle properties.

Aptamer-protein binding detected by asymmetric flow field flow fractionation.

Asymmetric flow field flow fractionation (AF4) should be suitable for the study of aptamer-target binding, because its gentle separation would impose little disturbance to the complex structure, and it can use carrier solutions with high salt concentrations to provide the most optimal interaction environment to the complex. However, no report has been found for such applications. Herein, we investigated the utility of AF4 as an effective tool for detection of the aptamer-protein complex. With the model system of human immunoglobulin E (IgE) and its aptamer, impacts on aptamer binding from the incubation and AF4 carrier solutions, as well as the flow conditions used during the sample focusing step, were studied. We found that the composition of the carrier solution, in particular, the presence of Mg(2+), strongly influenced the complex's integrity in AF4. Also, the focusing conditions during sample injection in AF4 affected the binding equilibrium. Our findings highlight the necessity of maintaining the optimal binding environment during the time course of complex measurement; and demonstrated the good compatibility of AF4 with salty buffers and its high simplicity in conducting on-channel incubation. With its capability to carry out size-based separation of analytes with a wide range of dimensions, AF4 can be employed for detection of large proteins and even biological particles using aptamers. AF4 is also valuable for study of aptamer-target binding under different buffer environments for better understanding of the structure-function relationship of aptamers.

Impact of carrier fluid composition on recovery of nanoparticles and proteins in flow field flow fractionation.

Flow field flow fractionation (F4) is an invaluable separation tool for large analytes, including nanoparticles and biomolecule complexes. However, sample loss due to analyte-channel membrane interaction limits extensive usage of F4 at present, which could be strongly affected by the carrier fluid composition. This work studied the impacts of carrier fluid (CF) composition on nanoparticle (NP) recovery in F4, with focus on high ionic strength conditions. Successful analysis of NPs in a biomolecules-friendly environment could expand the applicability of F4 to the developing field of nanobiotechnology. Recovery of the unfunctionalized polystyrene NPs of 199, 102, and 45 nm in CFs with various pH (6.2, 7.4 and 8.2), increasing ionic strength (0-0.1M), and different types of co- and counter-ions, were investigated. Additionally, elution of the 85 nm carboxylate NPs and two proteins, human serum albumin (HSA) and immunoglobulin (IgG), at high ionic strengths (0-0.15M) was investigated. Our results suggested that (1) electrostatic repulsion between the negatively charged NPs and the regenerated cellulose membrane was the main force to avoid particle adsorption on the membrane; (2) larger particles experienced higher attractive force and thus were influenced more by variation in CF composition; and (3) buffers containing weak anions or NPs with weak anion as the surface functional groups provided higher tolerance to the increase in ionic strength, owing to more anions being trapped inside the NP porous structure. Protein adsorption onto the membrane was also briefly investigated in salted CFs, using HSA and IgG. We believe our findings could help to identify the basic carrier fluid composition for higher sample recovery in F4 analysis of nanoparticles in a protein-friendly environment, which will be useful for applying F4 in bioassays and in nanotoxicology studies.

Advances in field-flow fractionation for the analysis of biomolecules: instrument design and hyphenation.

Field-flow fractionation (FFF) separates analytes by use of an axial channel-flow and a cross-field. Its soft separation capability makes it an ideal tool for initial fractionation of complex mixtures, but large elution volumes and high flow rates have limited its applicability without significant user handling. Recent advances in instrumentation and miniaturization have successfully reduced channel size and elution speed, and thus the volume of each fraction, making it possible to conveniently couple FFF with orthogonal separation techniques for improved resolution. More detailed analysis can also be performed on the fractions generated by FFF by use of diverse analytical techniques, including MS, NMR, and even X-ray scattering. These developmental trends have given FFF more power in the analysis of different types of molecule, and will be the direction of choice for further advances in FFF technology.

Cation exchange in ZnSe nanocrystals for signal amplification in bioassays.

ZnSe nanocrystals (NCs), possessing low native luminescence but high biocompatibility, were employed as labeling tags in bioassays. They were able to amplify each target recognition event thousands of times through a cation-exchange reaction (CXAmp) that released over 3000 encapsulated Zn(2+) from one single NC. The freed cations in turn triggered strong fluorescence from the Zn-responsive dyes. The present study demonstrated that CXAmp with ZnSe delivered superior detection performance in comparison to the conventional labeling methods. The overall fluorescence intensity of CXAmp using 5 nM ZnSe NCs was 30 times higher than that from 5 nM core-shell CdSe/ZnS quantum dots (QDs). The limit of detection (LOD) obtained with ZnSe-based CXAmp was 10-fold lower than with horseradish peroxidase (HRP) labeling, and the detection sensitivity, represented by the slope of the signal-versus-concentration curve, was 20-fold higher. When applied to detect immunoglobulin E (IgE) in a sandwich format, a LOD of 1 ng/mL was achieved. The highly sensitive CXAmp also allowed detection of the total IgE content in dilute human serum, in which the abundant matrix proteins exhibited less interference and more accurate quantification could be performed. Besides high signal amplification efficiency and good biocompatibility, CXAmp with ZnSe could be easily adapted to common laboratory settings and act as a universal labeling system for reliable detection of low-abundance targets.

Detection of microRNA by fluorescence amplification based on cation-exchange in nanocrystals.

Small RNA molecules are effective regulators of gene expression, and the expression signature of one subgroup of small RNA, the microRNA (miRNA), has been linked to disease development and progression. Therefore, detection of small RNA in biological samples will greatly improve the understanding of their functions and render effective tools to researchers for cellular process control and disease prevention. To solve the challenges in detecting the low-abundance and short strand-length of small RNA molecules, we designed a ligation-assisted binding assay and applied the cation exchange-based fluorescence amplification (CXFluoAmp) method developed in our group for detection. Nonfluorescent, ionic nanocrystals (NCs) of CdSe were conjugated to detection probes and immobilized onto the array surface via ligation with the target small RNA, miR21, which bound to the capture probe complimentarily. Each binding event induced by one target miR21 molecule was then amplified by the release of thousands of Cd2+ from one NC. The free Cd2+ immediately turned on the fluorescence of thousands of fluorogenic Rhod-5N molecules. With such a powerful signal amplification strategy, our assay achieved a limit of detection (LOD) of 35 fM and signals were detectable with analyte concentrations spanning over 7 orders of magnitude. We also identified the differential expression of miR21 in total RNA extracts from healthy breast tissue and diseased cells. Furthermore, our detection scheme demonstrated good specificity in small RNA detection, because significant signal intensity could be observed from small RNAs with one or two nucleotides difference in sequences. Thus, our assay has great application potential for disease diagnosis relying on miRNA biomarkers, or in small RNA expression profiling for new target discovery and functional study.