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Stanford University

Stanford Microfluidics Laboratory

Indirect Detection Using Mobility Markers

Principal Investigators: J.G. Santiago and Tarun Khurana

 

We have a developed a novel indirect detection assay that uses isotachophoresis and fluorescent species termed mobility markers to concentrate, separate, and detect non-fluorescent species. Isotachophoresis (ITP) has been used widely by analytical chemists as an electrophoretic separation technique and is also a robust sample preconcentration technique. ITP based indirect detection assay leverages a set of fluorescent moieties, termed mobility markers, whose electrophoretic mobilities bound those of non-fluorescent analytes of interest. Upon performing ITP based separation, the analytes and mobility markers segregate in order of reducing mobility between the leading (LE) and trailing electrolyte (TE). The non-fluorescent analytes are then detected as gaps between fluorescent marker zones. The length of the gap is a linear detector of the initial analyte concentration. We have demonstrated detection of amino acids, organic acids and phenols with detection sensitivity of up to 100 nM. 

In standard CE, ions are sensed directly measured by some detector sensitive to analyte ion properties such as fluorescence intensity, UV absorption cross-section, or electrochemical response.   Our ITP-based assay changes this paradigm entirely as we can:

 

  • Robustly, controllably, and repeatedly inject sample in disturbance-rich environments.

  • Simultaneously increase sample concentration by over 106 fold while we separate and detect.

  • Separate and then never loose fluorescence signal (since ion zones acquire a steady state profile).

  • Indirectly detect unlabeled, non-fluorescent ions with surrogate fluorescent molecules of our design.  This gives us both high sensitivity and specificity without sample treatment.

  • Provide unprecedented improvements in sensitivity, specificity, and dynamic range of analyte properties.

Figure 1 shows the Isotachophoretic mobility marker (IMM) assay where untreated sample is dispensed into the cathode reservoir of a single-channel microchip (or capillary) which is pre-filled with a stock solution of TE and pre-selected fluorescent mobility marker species.  The markers are a ladder of molecules with pH-dependent electrophoretic mobilities that bracket each of the sample species.  Upon application of an electric field, both the sample species and our fluorescent markers self-segregate into distinct, high concentration (up to 106 fold) zones between LE and TE.  The process creates a train of ions arranged by mobility where unlabeled sample analytes are detected unambiguously as “valleys” (or gaps) in the fluorescent marker signal. As shown by both our models and experiments, the known mobilities of fluorescent spacers bounding each analyte zone provide an upper and lower bound of analyte mobility. Further, the width of the “gap” between spacers is a linear measure of initial analyte concentration

 

 

Figure 1. Schematic of our ITP/mobility marker assay for injection, pre-concentration, separation, and indirect fluorescence detection of unlabeled analytes.

 

We have performed extensive IMM assay based separation and detection with a variety of non-fluorescent analytes such as amino acids, organic acids and phenols. In Figure 2, we present preconcentration, separation, and detection of two organic acids, acetic acid (ace) and 3-phenylpropionic acid (PP) using commercially available fluorophores Oregon Green carboxylic acid (OGCA), Fluorescein and Bodipy as mobility markers. We show the fluorescent mobility marker images at steady state for cases where acetic acid initial concentration was increased linearly from 12 µM to 48 µM, resulting in a linear increase in the gap width between spacers OGCA and Fluorescein.  This qualitatively confirms the linear relationship between the analyte zone length and its initial concentration.

 

Figure 2. CCD images of the fluorescent mobility markers in a 30 um microchannel demonstrating separation of acetate (Ace-) and phenylpropionate (PP) ions in four different experiments. The initial concentration of Ace- is increased linearly from 12 µM to 48 µM in (a)-(d) resulting in a linear increase in the spacing between fluorescent marker peaks 1 and 2.  PP concentration is held constant at 40 µM.  1: OGCA, 2: Fluorescein, 3: Bodipy

 

 

Image sequence for the separation and indirect detection of Acetate and phenylpropionate ions using three mobility markers.

 

In Figure 3, the length of acetic acid and phenylpropionic acid zones is plotted against their initial concentration ratio. Here, we varied the initial concentration of acetic acid from 12 mM to 48 mM and varied phenylpropionic acid initial concentrations from 20 mM to 80 mM. We quantitatively verify that the length of the unlabeled analyte zone is a linear measure of its initial concentration. Each data point represents three or four realizations per condition (error bars denote 95% confidence). The solid line is a linear fit to the experimental data with a regression coefficient of 0.98. The data are in good agreement with the theoretical prediction of the acetate and phenylpropionate zone lengths. We easily achieve a detection limit of 12 mMwith excellent repeatability.

 

Figure 3.  Length of the acetate and phenylpropionate analyte zones as a function of their initial concentrations for the low LE concentration experiment.  Solid line is a linear fit to the experimental data with regression coefficient of 0.98, and the dashed line is the theoretical prediction of LAceandLPP. LE was 5 mM Tris-HCl and TE was 5 mM sodium tetraphenylborate

 

Detection of Chemical Toxins

We recently completed a preliminary demonstration of IMMA in the fast and specific detection of four chemical toxins: four phenol derivatives.  Sample results are shown in Figure 3.  These toxins are all in the Environmental Protection Agency toxic release inventory of 581 chemicals.  All of these toxins have fully-ionized electrophoretic mobilities bounded between about 28 and 32 m2/V-s, but careful selection of the pH of the leading electrolyte allows us to easily distinguish between them.  This specificity is because ITP zones are very sensitive to both electrophoretic mobility and acid dissociation constants (pKa’s).

 


Figure 4.  IMMA microdevice results:  Simultaneous preconcentration, separation, and detection of four toxins:  2-Nitrophenol (NP), 2-Chlorophenol (CP), Trichlorophenol (TCP),  and p-Cresol (Cre).  All of these have fully-ionized mobilities which are within ~10%, but careful selection of assay pH helps leverage their differing pKa values for high resolution and specificity.  The markers here are Alexa Fluor 488, Oregon Green Carboxylic Acid, Fluorescein, Bodipy, and Green Fluorescent Protein (M1 through M5, respectively).  Note the quantitative relationship between signal gap width and analyte concentration (e.g., note the effect of doubling CP from the first to the second image).  Note the high/low/high signal intensity (digital encoding) scheme which we have integrated into the assay for unambiguous identification of analyte and marker zones.

 


Image sequence for the separation and indirect detection of four toxins using five mobility markers.

 

See related publications here