Rapid, Specific, and Efficient Affinity Purification of Target Molecules by Combining

Isotachophoresis and Affinity Chromatography

Viktor Shkolnikov and Juan G. Santiago

Department of Mechanical Engineering, Stanford University, Stanford, CA 94305, USA.

Affinity chromatography (AC) is a popular chromatographic technique for specific purification and/or analysis of proteins and nucleic acids from various samples [1]. However, when sample components of interest are present in low concentrations, a substantial volume of sample must be processed through the affinity substrate. Low target concentrations and high concentrations of fouling species in the sample also imply low target-probe binding rates [2]. These factors increase time of the affinity assay, can lead to poor substrate utilization, and/or poor purification yield. This limits applications of AC, especially in systems where system size and assay time are constrained.
We present a novel electrokinetic technique where we couple isotachophoresis (ITP) with AC to overcome these limitations of AC and achieve fast and selective purification with high column utilization. ITP simultaneously preconcentrates analytes and purifies them based on differences in electrophoretic mobility of sample components; ITP excludes species that may foul or compete with the target at the affinity substrate. ITP preconcentration accelerates the affinity reaction, reducing assay time, improving column utilization and allowing for capture of targets with higher dissociation constants. Furthermore, ITP-AC separates the target and contaminants into non-diffusing zones whose separation resolution increases linearly with time. Lastly, ITP-AC obviates the need for high pressure specialized pumps and directly integrates an automatic wash step into the process.
First, we will present an analytical model for spatiotemporal dynamics of ITP-AC. With this model, we identify and explore the effect of key process parameters including target distribution width and height, ITP zone velocity, forward and reverse reaction constants, and probe concentration on necessary affinity region length, assay time, and capture efficiency. Our model yields simple analytical relations for capture length and capture time in relevant ITP-AC regimes, and demonstrates how ITP greatly reduces assay time and improves column utilization. Next, we have completed and will present an experimental validation of our model with poly(glycidyl methacrylate-co-ethylene dimethacrylate) porous polymer monolith (GMA-EDMA PPM) functionalized with DNA probes inside a 500 µm diameter capillary. We demonstrated ITP-AC with 25 nt, Cy5 labeled DNA target and a DNA probe and studied the spatiotemporal dynamics using epifluorescence imaging. We chose to demonstrate ITP-AC with a DNA target as nucleic acids are important clinical markers and therapeutic agents [3-5] which often require rapid purification prior to analysis or use [6-8]. We varied the target concentration from 1 to
100 pg µl-1 and ITP velocity over the range of 10 to 50 µm s-1, and thereby explored over 4
orders of magnitude of scaled target amount. We observed very good agreement between predictions and experimental data for the spatiotemporal dynamics of the coupled ITP and
affinity process. Lastly, we purified 25 nt target DNA from 10,000-fold higher abundance background (contaminating) genomic fish sperm DNA. We performed this capture from over
200 µl sample volumes in under 1 mm column length and in less than 10 min, demonstrating especial relevance of this technique to miniaturized sample analysis systems.
In Figure 1a we show a schematic summarizing the ITP-AC assay. Figure 1b shows a spatiotemporal plot of experimentally measured target concentration in a typical ITP-AC experiment. Figure 2 shows quantitative spatiotemporal dynamics of target inside the affinity region as predicted by our model (top row) and experimentally measured (bottom row) for two target concentrations and two ITP velocities (three different conditions). Predicted spatiotemporal distributions of target agree very well with experiments over 3 orders of magnitude of scaled target amount (target amount scaled by the quotient of target velocity and the forward reaction rate constant). Lastly, Figure 3 shows spatiotemporal plots of sequence specific separation of rare 25 nt target DNA from 10,000-fold more abundant contaminating DNA using ITP-AC. We perform this purification in less than 10 min utilizing less than 1 mm column length.

Fig. 1. (a) Schematic of ITP-AC process. We filled the leading electrolyte (LE) reservoir and affinity column with LE buffer and place the sample and trailing electrolyte (TE) mixture into the TE reservoir (Step 1). We applied an electric field (from LE to TE) inducing ITP; the target species are extracted from the sample and focus into a sharp ITP peak (Step 2). The target migrates into the affinity region and is captured by the immobilized probe (Step 3). We then remove the LE and TE buffers and introduce a small slug of elution buffer to elute the target (Step 4). (b) Spatiotemporal plot of experimentally measured target concentration showing dynamics of a typical ITP-AC binding experiment. The concentrated target (visualized with Cy5 fluorescence) enters the porous affinity region from the left and is captured by the immobilized probes.

Fig. 2. Predicted (a, b, c) and measured (d, e, f) spatiotemporal dynamics of Cy5 labeled DNA target in ITP-AC inside the affinity region. The spatiotemporal plots show the logarithm of cross sectional area averaged fluorescence intensity of the target as a function of axial coordinate z and time. Location z = 0 is the leading edge of the PPM affinity region (c.f. Fig. 1). Predicted spatiotemporal distributions of target agree well with that experimentally observed for over 3 orders of magnitude of target amount scaled by the quotient of target velocity and the forward reaction rate constant.

Fig. 3. Experimentally measured spatiotemporal plots showing separation of rare target DNA from

10,000-fold more abundant contaminating fish sperm DNA using ITP-AC. The plots show the target and contaminant migrating from free solution into the porous polymer. (a) Fish sperm DNA (visualized via SYBR Green I) is not captured by the immobilized probe on the PPM and continues to migrate in ITP. (b) The Cy5-labeled, low-abundance target DNA of the correct sequence is quickly and selectively captured. (c) Overlapped signals from SYBR Green I and Cy5 optical channels show separation between trace target DNA and 10,000-fold more abundant contaminant in under 1 mm column length and in less than 10 min.

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