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Isotachophoresis-Based Surface Immunoassay Federico Paratore, Tal Zeidman Kalman, Tally Rosenfeld, Govind V. Kaigala, and Moran Bercovici Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 01 May 2017 Downloaded from http://pubs.acs.org on May 2, 2017
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Analytical Chemistry
Isotachophoresis-Based Surface Immunoassay Federico Paratorea,c, Tal Zeidman Kalmana,b, Tally Rosenfelda, Govind V. Kaigalac,||, and Moran Bercovicia,b* a
Faculty of Mechanical Engineering, Technion - Israel Institute of Technology, Haifa 3200003, Israel; bRussell Berrie Nanotechnology Institute, Technion –Israel Institute of Technology, Haifa, 3200003 Israel; cIBM Research – Zurich, Säumerstrasse 4, 8803 Rüschlikon, Switzerland ABSTRACT: In the absence of amplification methods for proteins, the immune-detection of low-abundance proteins using antibodies is fundamentally limited by binding kinetic rates. Here we present a new class of surface-based immunoassays in which protein-antibody reaction is accelerated by isotachophoresis (ITP). We demonstrate the use of ITP to pre-concentrate and deliver target proteins to a surface decorated with specific antibodies, where effective utilization of the focused sample is achieved by modulating the driving electric field (stop-and-diffuse ITP mode) or applying a counter flow that opposes the ITP motion (counterflow ITP mode). Using enhanced green fluorescent protein (EGFP) as a model protein, we carry out an experimental optimization of the ITP-based immunoassay and demonstrate a 1,300-fold improvement in limit of detection compared to a standard immunoassay, in a 6 min protein-antibody reaction. We discuss the design of buffer chemistries for other protein systems, and in concert with experiments provide full analytical solutions for the two operation modes, elucidating the interplay between reaction, diffusion and accumulation time scales, and enabling the prediction and design of future immunoassays. Surface-based immunoassays are the gold standard for specific protein detection, with extensive applications in all fields of life sciences, including clinical and point-of-care diagnostics, therapeutic drug monitoring, pharmacokinetics and drug development. Surface-based immunoassays can differ in terms of substrate (e.g., beads, microplates, etc.) and format (e.g., competitive, non-competitive, etc.); nevertheless, key to all is the binding between the target and the surface-immobilized antibodies (Abs), which enables separation of the protein from other undesired molecules in the sample. The antibody-protein complex is then sensed using a variety of methods, ranging from labeled detection antibodies1 (e.g., linked with enzymes, fluorophores, radioactive isotopes) to label-free sensors (e.g., cantilever2, optical micro-cavity3, nanowire4, nanoparticle5 and SPR6). In contrast to detection of low-concentration nucleic acids, in which molecular amplification methods7 (e.g., PCR8, LAMP9, etc.) exponentially increase the target concentration for fast and sensitive quantification, protein detection must rely solely on the original protein concentration in the sample. This represents a fundamental limit in detecting lowabundance targets, which is crucial for applications such as early disease diagnostic and analysis of single-cell contents10,11. Although a variety of signal-amplification methods exist (e.g., ELISA1 and its variants12, immunoPCR5,13), they improve only the transduction signal to noise ratio, and do not address the fundamental limitation of reaction kinetics, where the reaction time is inversely proportional to the target’s concentration. Electrokinetic focusing has recently been suggested as a way to increase the local sample concentration on top of a sensor to accelerate reaction kinetics. Han’s group used ion concentration polarization (ICP) for accelerating immune reaction, achieving a 500-fold improvement in R-PE immunebinding reaction14. This method has been parallelized and used to obtain a 65-fold improvement in the limit of detection (LoD) for detecting pre-labeled CRP over 30 min15, and a 500fold one in 1.5 h16. An alternative focusing method to ICP is
isotachophoresis (ITP), which does not require nano-to-micro channel interfaces or membranes, can be performed on cheap substrates (e.g., paper17,18) and can simultaneously be used as an effective sample-preparation method for extraction of targets from biological samples, including serum19–21, whole blood22 and cell lysates23. Karsenty et al.24 demonstrated the use of ITP for accelerated surface hybridization of DNA targets, achieving a 100-fold improvement. Han et al.25 demonstrated highly multiplexed surface reactions, showing a 4 orders of magnitude dynamic range of target concentration over an array of 60 spots. Moghadam et al. used ITP to enhance lateral flow assays, showing 100-fold improvement in limit of detection of Ab-Ab binding on a paper device18. Despite these potential benefits, the use of ITP for improving immunoassays remains largely unexplored. Arguably, this may be due to the higher complexity associated with the manipulation of proteins (as compared to, e.g., nucleic acids) and with predictions of their physicochemical properties. Immunoassays are known to be highly sensitive to the conditions of the buffer solution in which the reaction takes place, including pH, buffer composition, and ionic strength. At the same time, ITP requires the use of specific buffers that enable strong focusing. Here we present the first ITP-based surface immunoassay in which concentrated protein targets are delivered to an antibody-functionalized surface and detected by a fluorescent antibody, resulting in 1,300-fold improvement in LoD compared with a standard immunoassay. To the best of our knowledge, this is the largest enhancement in the LoD achieved to date using accelerated reaction. This enhancement is enabled by two operation modes that allow a longer reaction time by stopping the ITP plug over the reaction site either by turning off the electric field or by applying a counter-pressuredriven flow. We investigate the effect of the buffer system on the performance of both the ITP and the immunoassay, and provide buffering conditions that simultaneously optimize the ITP focusing and the primary binding reaction. Using
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enhanced green fluorescent protein (EGFP) as the target
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protein, we
Figure 1. Schematics and raw experimental images of the immunoassay principle. a, ITP focuses proteins of interest and delivers them to the reaction site. b, The accelerated immunoreaction occurs between the capture antibodies bound to the surface and the proteins. We distinguish between three operation modes: b1, Pass-Over ITP (PO-ITP): the focused proteins react with the surface while electro-migrating downstream; b2, Stop and Diffuse (SD-ITP): the electric field is temporarily stopped, enabling a longer reaction time while proteins diffuse away from the reaction site; b3, Counterflow ITP (CF-ITP): the ITP plug is kept over the reaction site by a pressure-driven counterflow, enabling a longer reaction time while keeping the proteins focused. c, The final immuno-detection step is based on labeled (fluorescent, enzyme, etc.) antibodies binding to the reacted targets. reduce the limit of detection on a standard microscopy system from 300 pM to 220 fM. Alongside the experimental demonstration, we present new and experimentally validated analytical models that describe the physics of each mode and identify key parameters governing the reaction in these regimes, allowing the prediction and optimization of such assays. Principle of the ITP-based immunoassay In peak-mode ITP, analytes focus at the sharp electric field gradient between a leading electrolyte (LE) and a trailing electrolyte (TE), containing high-mobility leading ions (L) and low-mobility trailing ions (T), respectively26. The electroneutrality in the bulk is ensured by counterions (CI), which have a net charge opposite to that of T- and L-ions. The sample can be mixed with the L- or T-ions (continuous injection) or be initially placed between the LE and TE zones (finite injection). In this work, we will consider the case where proteins are negatively charged (anionic ITP) and initially placed in the well containing the T-ions and continuously enter the channel, accumulating at the ITP interface. The analysis can easily be extended to other modes of sample injection and to positively charged proteins (cationic ITP). Herein, we adopt a mathematical notation that specifies the ion in the subscript and the zone in the superscript. In the steady state, the L- and T-ions in their respective zones move with the same ITP velocity VITP = µTTE E TE = µ LLE E LE , where µ and E indicate
the electrophoretic mobility and the electric field, respectively. As illustrated in Figure 1a, we consider a straight channel with height ݄ containing a reaction site of length ws placed at a distance xs from the TE reservoir and functionalized with capture Abs (cAbs) at a surface density bm . We define the ycoordinate perpendicular to the channel with y = 0 coinciding with the reaction site at the bottom channel wall. Far from the reaction site, at a distance xitp (t ) from the channel entrance and for negligible dispersive effects, the analyte concentration can be approximated as a Gaussian24,27, 2 c( x, y, t ) = α c0w exp[−8( x − xitp ) 2 / witp ], (1) where c0w is the initial protein concentration in the TE well,
α = 8 / π xs / witpηT is a dimensionless pre-concentration factor and ηT is the accumulation factor given by17,24,28
µaTE − µTTE µaw σ LE TE w , LE µL µa σ
ηT =
(2)
where µa is the electrophoretic mobility of the analyte. The width of the ITP interface, witp , depends on the buffer composition and dispersion29 and cannot be accurately
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Analytical Chemistry
predicted analytically; therefore, throughout this work, we use its value as obtained from experiments. As the ITP interface reaches the reaction site, i.e., the Gaussian overlaps with the reactive surface, the focused proteins start binding to the surface. Assuming a second-order reaction30, the binding rate is given by
x − xs ∂b = Π δ ( y ) kon ( bm − b ) c − koff b , ∂t ws
(3)
where b [mol m −2 ] is the surface concentration of bound proteins. Π is the top-hat function
Π ( ( x − xs ) / ws ) = H ( x − xs + ws / 2) − H ( x − xs − ws / 2) ,
H is the Heaviside function, and δ ( y ) is the Dirac delta function, indicating that the reaction occurs only between x = xs − ws / 2 and x = xs + ws / 2 at the bottom surface. kon [M -1 s-1 ] and koff [s-1 ] are the reaction on- and off-rate, respectively. This equation must be solved in coupling with the evolution of the analyte concentration in the bulk, which can be described by the Nerst–Planck equation,
∂c ∂ ∂c ∂ ∂c + ( µa E + vPD ) c − D + D = ∂t ∂x ∂x ∂y ∂y
(4)
x − xs −Π δ ( y ) kon ( bm − b ) c − koff b ws where vPD ( y ) is the pressure-driven flow velocity in the xdirection, and D is the molecular diffusivity of the protein. As illustrated in Figure 1, the different flux terms in equation (4) introduce three distinct regimes in which ITP focusing can be used to accelerate the binding rate and hence the immunoassay signal. In the following subsections, we analyze each of these regimes and compare their predicted performance with that of a standard immunoassay. Results Pass-Over ITP (PO-ITP). The simplest mode of operation is one in which the focused proteins continuously electro-migrate in the channel, and react with the functionalized surface only for a short period of time during which they spatially overlap. This case has been investigated by Karsenty et al.24 and Han et al.25. For completeness and consistency, we repeat their main assumptions and findings here, but refer the reader to their papers for complete details. The characteristic diffusive timescale along the height of 2 the channel is given by τ Dy = h / D , whereas the characteristic advection time of the focused sample over the surface is τ PO = witp / Vitp . As long as τ PO < τ Dy , the bulk concentration along the y-axis can be considered constant, and equation (4) can be depth-averaged and simplified to
∂ca ∂ ∂c + µa Eca − D a = ∂t ∂x ∂x x − xs 1 −Π kon ( bm − b ) ca − koff b ws h
,
(5)
1 h cdy is the depth-averaged concentration, and h ∫0 we have set vPD = 0 because no pressure-driven flow is
where ca =
−4 imposed. For values of witp = 10 m , VITP = 50 ⋅10−6 m s−1 ,
D = 7.8 ⋅ 10 −11 m 2s −1 31, typical in our experiments, we obtain τ PO ~ τ Dy , which indicates the limit of applicability. Here, we neglect any dispersion because the focused plug motion is characterized by small Péclet numbers and the effective diffusivity can be approximated by the molecular one27,29. As shown by Karsenty et al.24, the concentration at the ITP interface does not change significantly during the short reaction time, and thus the concentration profile (equation (1)) can be assumed constant in time. Substituting equation (1) into equation (3) and integrating over the transition time of the sample over the interface ( τ PO ), the expression for the fraction of bound sites is obtained:
bPO f α c0w = 1 − exp −kon ( f α c0w + K d )τ PO , bm f α c0w + K d
(
)
(6)
where bPO is the number of target proteins bound to the surface in the PO-ITP mode,
f = π 8erf
geometrical factor, and K d = koff kon
( 2)
is a
is the dissociation
constant. ITP focusing has a direct impact on the characteristic reaction timescale τ r = 1 / kon ( f α c0w + K d ) . The higher the accumulation, the faster the reaction, i.e., the lower the timescale τ r . This is particularly beneficial for concentrations in the range K d f α < c0w < K d , in which ITP boosts the kinetics from a slower regime, dominated by the off-rate τ r = 1/ koff , to a faster one, dominated by the on-rate
τ r = 1 / kon f α c0w . However, this process does not realize the full potential of ITP focusing as the overall time spent by the protein plug on the surface allows only ~1% of the proteins to react with the surface. The majority of the sample continues electro-migrating with the ITP interface and is removed from the reaction site. Stop and Diffuse ITP (SD-ITP). The reaction efficiency can be further improved by stopping the driving voltage and allowing the focused sample to react with the surface for any desired length of time. However, as the electric field is turned off, the sample diffuses away, resulting in gradual decrease in bulk concentration over the reaction site; therefore, the effect of the assay time on the signal must be understood. We identify a characteristic depletion timescale, τ dep = h / bm kon , on which the reaction reduces the delivered
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sample bulk concentration by approximately e −1 of its initial value. As long as the sample can be assumed well mixed along the height of the channel, i.e., the diffusion along the y-axis is faster than the reaction at the surface, τ Dy
