Anal. Chem. 2008, 80, 808-814
Controlling Data Quality and Reproducibility of a High-Sensitivity Immunoassay Using Isotachophoresis in a Microchip C. Charles Park,*,† Irina Kazakova,† Tomohisa Kawabata,‡ Michael Spaid,§ Ring-Ling Chien,‡ H. Garrett Wada,‡ and Shinji Satomura‡
Caliper Life Sciences, Mountain View, California 94043, Wako Pure Chemical Industries, Mountain View, California 94043, and Cambrios Technologies, Mountain View, California 94043
This report describes a method of controlling the sensitivity and reproducibility of a microchip-based immunoassay by using isotachophoresis to preconcentrate the antigen and antibody prior to binding. Gel electrophoresis separation is coupled to the preconcentration step to separate the immunocomplex products formed. The system employs a quartz-based LabChip that automates the metering, preconcentration, reaction, separation, and detection. The system also uses a handoff mechanism that switches the immunocomplex from the stacking mode to the separation mode. We show that the handoff timing affects the data quality and repeatability of the electropherograms, and we demonstrate an automatic handoff mechanism to precisely control the signal intensity and separation of peaks of interest. In so doing, the automatic handoff mechanism also improves the reproducibility of the assay. When applied to the homogeneous liquid-phase detection of r-fetoprotein, a common tumor marker, the system shows a greater than 200-fold stacking of specific analytes of interest. Due to their selectivity and sensitivity, immunoassays have long been used in clinical diagnostics and have made significant scientific and commercial impact. Originating from the pioneering work of Yallow and Berson,1 Enzyme-linked immunosorbent assay (ELISA)2 is the most commonly used immunoassay format. While it is highly sensitive, the heterogeneous nature of ELISA requires a series of steps like mixing, incubation, and washing that may take hours to perform. It also requires antibodies with high affinities, in order to maintain stringent washing conditions and low background. A more recently developed capillary electrophoresis-based immunoassay (CEIA) simplified many of the steps and lowered the time per assay by allowing reactions to occur more rapidly in the solution phase. CEIA also offers reduced * To whom correspondence should be addressed. E-mail: Charles.Park@ CaliperLS.com. † Caliper Life Sciences. ‡ Wako Pure Chemical Industries. § Cambrios Technologies. (1) Yalow, R. S.; Berson, S. A. Nature 1959, 184, 1648-1649. (2) Engvall, E.; Perlmann, P. J. Immunochem. 1972, 109,129-135.
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sample and reagent consumption.3,4 Among the technical challenges that still need to be solved for CEIA are lower throughput and a requirement for optimization of separation. Another development in immunodiagnostics that addressed some of the fore-mentioned issues as well as reduced assay time was the advance in micromachining that gave rise to the lab-ona-chip technology. Most of the recent immunoassays on a microchip, however, used solid-phase immunocapture. To overcome the sensitivity limitation, preconcentration with antibodyfunctionalized microbeads as well as surface modifications to improve antibody binding has been implemented.5-8 Although the use of microbeads does push the sensitivity limit to picomolar and even femtomolar ranges, the microbead format still requires relatively long assay times and the need for high-affinity antibodies limits the range of potential analytes.9 By combining the CEIA format with lab-on-a-chip technology, it is possible to reduce reaction time, allow the use of ligands with lower affinities, and incorporate automation of many labor-intensive steps. Throughput also can be improved by using microchips with multiple channels. Chiem et al.10 were among the first to demonstrate liquid-phase automated immunoassay on a microchip. While liquid-phase immunoassays based on lab-on-a-chip technology offer high turnaround, low sample volume consumption, and simplicity of operation, the sensitivity limit still remains to be an area of great challenge. Several different attempts such as improvements of detection systems,11 enhancement of fluorescence,12,13 and preconcentration14-16 were made to improve the (3) Nielsen, R. G.; Richard, E. C.; Santa, P. F.; Sharknas, D. A.; Sittampalam, G. S. J. Chromatogr. 1991, 539, 177-185. (4) Yeung, W. S.; Luo, G. A.; Wang, Q. G.; Ou, J. P. J. Chromatogr., B 2003, 797, 217-228. (5) Haes, A. J.; Terray, A.; Collins, G. E. Anal. Chem. 2006, 78, 12-20. (6) Philips, T. M.; Weilner, E. J. Chromatogr., A 2006, 1111, 106-111. (7) Bai, Y.; Boreman, M.; Juang, Y.; Tang, I.; Lee, L.; Yang, S. Langmuir 2006, 22, 9458-9467. (8) Koskinen, J.; Meltola, N.; Soini, E.; Soini, A. Lab Chip 2005, 5, 1408-1411. (9) Bange, A.; Halsall, H. B.; Heineman, W. R. Biosens. Bioelectron. 2005, 20, 2488-2503. (10) Chiem, N. H.; Harrison, D. J. Clin. Chem. 1998, 44, 591-598. (11) Sowell, J.; Parihar, R.; Patonay, G. J. Chromatogr., B 2001, 752, 1-8. (12) Wang, Y.; Su, P.; Zhang, X.; Chang, W. Anal. Chem. 2001, 73, 5616-5619. (13) Koizumi, A.; Morita, T.; Murakami, Y.; Morita, Y.; Sakaguchi, T.; Ykoyama, K.; Yamiya, E. Anal. Chem. Acta 1999, 399, 63-68. (14) German, I.; Kennedy, R. T. Anal. Chem. 2000, 72, 5365-5372. (15) Tsukagoshi, K.; Nakamura, T.; Nakajima, R. Anal. Chem. 2002, 74, 41094116. 10.1021/ac701709n CCC: $40.75
© 2008 American Chemical Society Published on Web 01/04/2008
detection limits. In particular, Herr et al.17 described preconcentration with a photopolymerized cross-linked polyacrylamide gel barrier within a microfluidic device. Also, Mohamadi et al. used transient isotachophoresis and capillary gel electrophoresis to stack and separate the antigen-antiboy complex.18 We developed a system that increases the sensitivity of a microchip-based immunoassay using isotachophoresis (ITP) coupled to gel electrophoresis (GE). Based on the pioneering work of Kohlrausch,19 ITP makes use of a discontinuous buffer system to concentrate and separate ions.20 Though it can be used as a standalone system, ITP alone usually does not provide enough separation between stacked zones to resolve the separated ions of interest for systems with trace amount of analytes. A common solution to such limitations of ITP is to couple a homogeneous buffer system for separating a group of stacked peaks concentrated by ITP.21 Such coupling of the ITP stacking and separation was demonstrated on a microchip recently.22,23 We applied the microchip that uses ITP and GE to an immunoassay that requires an immunoreaction of precisely metered reagents. By using microchip metering of reagents, immunoassay reaction, preconcentration, separation, and detection were integrated into a single LapChip system. Furthermore, the system allows the users to control the data quality, namely, peak separation and intensity, without changing assay format or chip architecture. The data quality is controlled mainly by controlling the time at which the system switches from the ITP step to the GE step. Furthermore, by implementing a computer-controlled switch mechanism, the repeatability of the assay was improved. EXPERIMENTAL SECTION Reagents and Hardware. In order for ITP with anions to work properly, the choice of leading and trailing electrolytes must be made in such a way that both electrolytes have enough buffer capacity to resist pH changes during the electrophoresis involved in the preconcentration and separation steps. Also, the major current-carrying anions in the leading and trailing buffers must have mobilities that are higher and lower, respectively, than the anionic complexes of interest. The major current carriers in the leading buffer were tris(hydroxymethyl)aminomethane chloride (TRIS-Cl) and NaCl. The major current carriers in the trailing buffer were tris(hydroxymethyl)aminomethane (TRIS) and N-2hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES). Both the ITP and GE steps were carried out in dilute solutions of poly(2-dimethylaminoethyl methacrylate) (pDMA). All the regaents were purchased from Sigma unless noted otherwise. The leading buffer composition was 0.9% pDMA (Polysciences) (w/v), 3% glycerol; 75 mM TRIS-Cl pH 8; 50 mM (16) Wang, Q.; Luo, G.; Ou, J.; Yeung, W. J. Chromatogr., A 1999, 848, 139149. (17) Herr, A.; Throckmorton, D.; Davenport, A.; Singh, A. Anal. Chem. 2005, 77, 585-590. (18) Mohamadi, M.; Kaji, N.; Tokeshi, M.; Baba, Y. Anal. Chem. 2007, 79, 36673672. (19) Kohlrausch, F. Ann. Phys. Chem. 1897, 62, 209-239. (20) Krivankova, L.; Pantuckova, P.; Bocek, P. J. Chromatogr., A 1999, 838, 5570. (21) Everaerts, F.; Vherhegeen, T.; Mikkers, F. J. Chromatogr. 1979, 169, 2138. (22) Walker, P.; Morris, M. Anal. Chem. 1998, 70, 3766-3769. (23) Wainright, A.; Williams, S.; Ciambrone, G.; Xue, Q.; Wei, J.; Harris, D. J. Chromatogr., A 2002, 979, 69-80.
NaCl; 0.01% albumin from bovine serum; 0.05% Tween-20; and the trailing buffer composition was 0.9% pDMA (Polysciences) (w/ v), 3% glycerol; 75 mM TRIS; 0.01% BSA; 0.05% Tween-20; 125 mM HEPES. The sample was introduced in a sample buffer with 0.9% pDMA (Polysciences) (w/v), 3% glycerol; 75 mM TRIS-Cl pH 8; 50 mM NaCl; 0.01% BSA; 0.05% Tween-20. AFP L1 and fluorecently labeled DNA-coupled antibody (WA1) were produced by Wako Pure Chemical Industries Ltd. (Osaka, Japan) as described in detail by Kawabata et al.24 The antibody was diluted to final concentration of 20 nM in leading buffer, and 5 nM AFP L1 was spiked into normal human serum (Sigma) and diluted 1:10 in sample buffer. The reaction between AFP and WA1 is as follows:
AFP + WA1 T AFP-WA1
(1)
With the concentrations of AFP and WA1 used, the equilibrium is driven to covert most of the AFP into the AFP-WA1 complex with the majority of the WA1 left unbound. The microchips used for this study were made by Caliper Life Sciences from their manufacturing facilities in Mountain View, CA. The chips were made by thermally bonding a quartz plate with microfluidics channels isotropically etched on one side to another plate with holes drilled to act as reagent wells. The bonded plates were glued to an acrylic-based caddy that acts as an interface between the instrument and chip.25 The channels were washed with 1 N NaOH and thoroughly rinsed with water prior to each experiments. The pressure and voltage were controlled using a Caliper42 and a modified version of a LabChip90, both of which were manufactured by Caliper Life Sciences.25 Caliper42 is a customizable microfluidics-based electrophoresis system capable of independent voltage and pressure control. For detection, the Caliper42 is equipped with the photomultiplier detection system 814 (Photon Technology International) on a Nikon TE300 microscope with a 35-mW laser diode (Hitachi 635 nm) filtered through a HYQ Cy5 epifluorescent filter combination filter cube (Nikon). The LabChip90 is an automated microfluidics-based electrophoresis system capable of analyzing samples from 96- and 384-well microtiter plates. For the purposes of the current study, the LabChip90 was modified to allow independent pressure, voltage, and current controls of all of the pressure and voltage nodes. The optics block in the LabChip90 is made up of a 35-mW laser diode (Hitachi 635 nm), custom-made filter set with excitation at ∼630 nm and emission at ∼700 nm, and a single element photodiode (Hamamatsu). Signal processing in both the Caliper42 and LabChip90 is handled by a customized software that tracks continuous signal intensity as a function of time. Chip Architecture and Operation. As described in the Experimental Section, ITP requires that trailing and leading buffers with lower and higher mobilities, respectively, envelope the sample of interest. The chip architecture used for this study establishes three zones: trailing, sample, and leading buffers. Figure 1A shows a schematic diagram of the chip architecture and the zones established by the chip. The lengths of the trailing, (24) Kawabata, T.; Watanbe, M.; Nakamura, K.; Satomura, S. Anal. Chem. 2005, 77, 5578-5582. (25) Henry, C. S., Ed. Microchip Capillary Electrophoresis Methods and Protocols; Methods in Molecular Biology 339; Humana Press: Totowa, NJ, 2006; pp 129-143.
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Figure 1. Schematic illustration of the chip architecture and operation. (A) Chip is loaded with trailing and leading buffers surrounding the sample by applying vacuum to wells 4 and 5. Reactants A represents the Alexa-labeled-140-bp DNA-coupled-WA1, and reactant B represents the AFP. Reactants A and B react during the loading process to produce the sample product in the sample zone. (B) ITP stacking initiated by applying an electric field between wells 1 and 7. The voltage at well 6 is controlled to maintain a zero current through well 6 during the ITP process and is monitored for triggering purposes. (C) When the stacked sample reaches the junction for ITP-GE handoff, the cathode is switched from well 1 to well 6. At this point, the voltage at well 6 reaches the maximum value. (D) With well 6 as the cathode, GE separates the product AB and unreacted reactant A.
sample, and leading zones as shown in Figure 1A are 10, 10, and 43 mm, respectively. The length of the channel connecting well 6 to well 7 is 26 mm. All the channels are isotropically etched with depth of 15 µm, bottom width of 80 µm, and top width of 110 µm. Well 1 contains the trailing buffer; well 2 contains one of the reactants in this case the Alexa Fluor 647 (Invitrogen Catalog No. A-20006) labeled-140-bp DNA coupled WA1; well 3 contains the second reactant, in this case the AFP; well 4 and well 5 contain small amounts of the sample buffer. Wells 6 and 7 contain the leading buffer. By applying vacuum of ∼5 psi to wells 4 and 5 in Figure 1A, while keeping all the other wells at atmospheric pressure, the three zones mentioned can be established in the chip. At the same time, the three zones are being established, the reaction between reactant A and reactant B proceed in the mixing channel designated as “A + B” in Figure 1A. Because the amounts of A and B that react are controlled by the applied pressure and the relative channel resistance of the chip, the metering of the reactant is controlled by the microchip architecture and the operating parameters of the system. Once the three zones have been established, ITP is initiated by applying a voltage difference to the chip as shown in Figure 1B. The “-” and “+” designate the voltage difference, while the “V” in Figure 1B signifies an automatic voltage control in well 6. The applied voltage difference for ITP was ∼2900 V. The automatic voltage control in well 6 adjusts the voltage applied to well 6 to ensure that the current passing through well 6 is controlled to 810
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zero during the time the voltage drop is applied between well 1 and well 7. The value, V, represents the voltage value required to maintain a zero current through well 6, which is also equivalent to the actual voltage at the junction without the side channel. The voltage V versus time plot in the lower right-hand corner of Figure 1B shows the voltage signal response of the control scheme as ITP progresses. Note that V increases with time. Since the trailing buffer had lower conductivity than the leading buffer in the buffer system used, the motion of the trailing buffer-leading buffer interface toward the anode effectively increased the overall electrical resistance of the channel connecting well 1 to well 6. Such an increase in the electrical resistance requires a higher voltage in well 6 to maintain a zero current condition, effectively increasing the value of V as a function of time. The voltage at well 6 continues to increase with time until the trailing buffer-leading buffer interface arrives at the handoff junction where the channel leading out of well 6 meets the main channel connecting the anode and the cathode as shown in Figure 1C. Once the trailing buffer-leading buffer interface passes the junction, the resistance in the section upstream of the handoff junction remains constant while the total resistance continues to increase as more and more low-conductivity trailing buffer is injected into the channel. Based on the physics of the voltage divider, the consequence is a drop of the voltage at the handoff junction. Furthermore, as shown in Figure 1C, the sample introduced to the sample zone initially is stacked in between the
Figure 2. (A) Typical electropherogram obtained after preconcentration. The quantity I represents the signal intensity; the quantity S represents the peak separation. (B) Fluorescence signal of the sample without preconcentration or separation. The quantity i represents the signal intensity of the unstacked sample. The cutout shows a schematic illustration of how the samples from wells 2 and 3 are directed to the detector using multiport pressure control without any regent from wells 1 or 4-6 leaking to the detector.
trailing and leading buffers and occupies a shorter length in the chip after the preconcentration step. At the point where the voltage in well 6 reaches a maximum value, the cathode is switched from well 1 to well 6 so that the voltage difference is established between well 6 and well 7 as shown in Figure 1D. Since buffers both upstream and downstream of the sample in this configuration are leading buffer, the system switches from ITP in Figure 1C to GE in Figure 1D. The applied voltage difference for GE was ∼2100 V. After the switch, the sample ions that are stacked in Figure 1C begin to separate based on their mobilities as shown in Figure 1D. By placing a detector near the end of the separation channel, the separated components of the sample are shown as separate peaks of an electropherogram. The voltage reading on well 6 shows an abrupt drop when the switch is made and stays at a constant voltage commanded for the separation. The switch made between Figure 1C and Figure 1D represents a handoff from ITP to GE. One additional advantage of this switch scheme is that any fluorescent species downstream of the sample with lower mobilities are left behind out of the separation channel, decreasing assay background fluorescence. RESULTS AND DISCUSSION A typical electropherogram obtained from the process described in the Chip Architecture and Operation section is shown in Figure 2A. There are two prominent peaks in the electropherogram shown. The larger peak that arrives at ∼83.75 s is the unreacted conjugate while the smaller peak that arrives at ∼84.5 s is the AFP immune complex. For the purposes of evaluating the electropherogram, peak intensity, I, and peak separation, S, are measured as shown in Figure 2. Peak intensity is measured as the peak height of the major peak. Peak separation is the difference in the arrival times of the centers of the two peaks of interest. Stacking Factor. The sensitivity of a detection system is affected by many different factors such as assay format, instrument, and users. In this study, we focus on the effect of ITP preconcentration on sensitivity by reporting the stacking factor. The stacking factor for the preconcentration method presented in this study was estimated by comparing the signal intensity, I, to the signal intensity of the sample without preconcentration. The
cutout in Figure 2B shows a schematic illustration of how the signal intensity of the unconcentrated sample was measured. Using multiport pressure control, samples from wells 2 and 3 can be directed toward the detector without any reagent in wells 1, 4, 5, or 7 diluting the sample signal. The data plotted in Figure 2B show the signal obtained from the pressure condition shown in the cutout. The signal from ∼350 to ∼400 s in Figure 2B represents the baseline signal of just the leading buffer from well 6. The elevated signal at ∼500 s represents the signal of the samples from wells 2 and 3 being directed toward the detector. The depressed signal from ∼550 to ∼650 s represents the baseline signal of the leading buffer from well 6 replacing the sample signal from wells 2 and 3. The quantity, i, in Figure 2B is obtained by taking the intensity difference between the average signal intensity of the baseline and that of the sample. The quantity, i, represents the signal intensity of the unstacked sample. The stacking factor is then simply calculated by the ratio
stacking factor ∼ I/i
(2)
Using the above equation, the stacking factor for the system being presented is calculated to be ∼200. Signal Intensity and Peak Separation. While the stacking factor indicates how much signal increase the system is capable of producing during the ITP stages of the system, peak separation indicates how well the GE is able to separate the components of interest from other comigrating species in the sample. Especially when the sample contains species of interest and comigrating species whose mobilities are closely matched, an effective GE separation becomes vital to the usefulness of the system. For the current system, where ITP and GE are coupled in a microchip, the signal strength, I, and peak separation, S, are not independent of each other. Figure 3A shows four example electropherograms that illustrate the interdependency of I and S. Run A1 in Figure 3A shows what appears to be two peaks around 81.75 s that are essentially collapsed into one peak with a shoulder peak, while run A4 shows two baseline separated peaks at ∼83.5 s. The two peaks are the unreacted the WA1 and AFP-WA1 complex peaks as described in Figure 2. Runs A2 and A3 show peak separations that are somewhere in between those of runs A1 and A4. An Analytical Chemistry, Vol. 80, No. 3, February 1, 2008
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Figure 3. (A) Electropherograms of four different runs that have different delay times resulting in different peak intensity and separation. (B) Closeup views of the handoff voltages near their handoff voltage of a run with a long delay time resulting in run A1 of Figure 3A. (C) Handoff voltage of a run with a short delay time resulting in run A4 of Figure 3A.
electropherogram resembling run A1 would pose severe problems, if quantitation of the smaller AFP immune complex is necessary. Also, note that the signal intensity, I, of run A1 is greater than that of run A4 and those of runs A2 and A3 are somewhere in between those of A1 and A4. The experimental results shown in Figure 3A suggest that I and S are inversely related. The mechanism for such an interdependency between I and S may be explained by recalling that the ITP-GE system chosen uses a handoff mechanism that relies on a handoff from ITP to GE at the moment that the stacked sample reaches the handoff point. Refer to the explanation of Figure 1 in the Chip Architecture and Operation section of this report for an explanation of the handoff mechanism. If the handoff occurs exactly at the moment where the stacked sample is at the intersection, as shown in Figure 1C, then no trailing buffer can enter the separation channel. Without any trailing buffer in the separation channel, GE may be carried out immediately following the handoff to maximize the peak separation. However, allowing the ITP to continue beyond the point depicted in Figure 1C would prolong the advancement of the trailing buffer-leading buffer-interface into the separation channel, causing the trailing buffer to enter the separation section of the chip. If trailing buffer penetrates into the separation channel, then GE separation cannot begin immediately after handoff. Rather transient isotachophoresis begins where ITP continues until the leading buffer behind the trailing buffer overtakes the trailing buffer that has penetrated the separation zone. Once the sample of interest is enclosed by leading buffers on either end, GE begins to separate the individual components in the stacked sample. If 812
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no trailing buffer enters the separation channel, the electropherogram shows larger peak separation with lower peak intensity. The greater the amount of trailing buffer that enters the separation channel, the smaller the peak separation, S, and higher the peak intensity, I, are in the resulting electropherogram. One method of gauging the extent to which the trailing buffer has penetrated into the separation channel is by measuring the time the system spends in ITP state after the trailing bufferleading buffer interface has passed the intersection of the stacking and separation sections of the chip. Figure 3B shows how this time, referred to as the delay time, may be measured. Figure 3B shows a closeup view of the voltage read from well 6 during the ITP step. Near its maximum point, the voltage trace of well 6 shows two prominent kinks labeled as “t1” and “t2” in Figure 3B. Experiments show that the time position of the trailing bufferleading buffer interface coincides with the time position of the kink t2. Hence, the time interval between kink t2 and the handoff point labeled as t3 in Figure 3B represents the effective delay time labeled as “∆t”. In agreement with the mechanism proposed, Figure 3B shows that run A1 with longer ∆t produces an electropherogram with higher I and smaller S, while Figure 3C shows that run A4 with shorter ∆t produces an electropherogram with lower I and larger S. A more comprehensive sweep of ∆t and its effects on peak intensity and separation shows a validation of the proposed mechanism. Figure 4 shows the effect of delay time on peak intensity (I) and peak resolution (R) defined as the peak separation S divided by the full width at half-maximum of the major peak.
Figure 4. Dependence of experimentally obtained signal intensity (I) and peak resolution (R) on delay time (∆t) plotted against the corresponding scaling predictions.
Note that the experimentally obtained I increases with ∆t while R decreases with ∆t. Figure 4 also shows the scaling dependence of I and R on ∆t based on the capillary electrophoric separations model presented by Bharadwaj et al.26 The scaling results plotted take into account the effect of changing the time of separation on I and R as a result of changes in ∆t in the axial diffusion limit. Note that the scaling predictions underpredicts the magnitude of the effects of ∆t on I and R, suggesting that the trailing buffer that enters the separation zone plays an important role in the separation process. The results also suggest that, depending on the assay conditions such as the mobilities of the species involved or conductivities of the buffers used, the absolute values of I and R may vary significantly. However, the trends observed and the mechanism explained may be used to tune the chip for the data quality desired. For instance, if the sample being analyzed requires more sensitivity than resolution, then ∆t can be increased until the desired values of I and R are obtained. In conjunction with other assay parameters, the delay time allows users to control the data quality without altering the chip geometry or assay conditions. Repeatability. Note that in Figure 3B and C the difference between a highly resolved and poorly resolved sample separation is the difference between a ∆t of 1 s or ∆t of 1/2 s. Furthermore, Figure 4 shows that a 1/2 s difference in the delay time may show up to a 2-fold difference in I. Consequently, the ability to trigger the handoff with a fraction of a second accuracy is the key to making sure that the interdependence of I and S is controlled. Unfortunately, slight changes in concentrations of components or manufacturing defects introduce enough arrival time variations to make a significant difference in the data quality. For example, the first kink labeled t1 in Figure 3B appears ∼1/2 s slower than the corresponding first kink in Figure 3C, even though the operating conditions up to the first kink in the two experiments are theoretically identical. In order to ensure that unintended differences in ITP traverse time do not significantly affect the data quality of interest, a handoff mechanism that uses a repeatable handoff time (Th) was em(26) Bharadwaj, R.; Santiago, J.; Mohammadi, B. Electrophoresis 2002, 23, 27272744.
Figure 5. Electropherograms of four different runs with a computercontrolled delay time to maximize separation and repeatability. The numbers next to the run designations represent the peak heights of the AFP complex peak for that run.
ployed. The mechanism involved the computer monitoring the voltage signal from well 6 to recognize the kinks such as t1 and t2 in Figure 3B. The kinks are detected by measuring the change of voltage as a function time. The change of voltage is defined as n
∑
∆V )
n/2
∑V
Vi
n/2+1
n/2
-
i
1
n/2
(3)
In the above equation, ∆V is the change of voltage from well 6, V is the voltage, and n is the number of data points interrogated. A kink is defined as a point where ∆V value changes. A reference time point for the kink (Tk) was defined as the time at which ∆V ) -0.5 with n ) 10. A user-defined delay time (∆t) was added to Tk to determine the time for handoff (Th) as follows:
Th ) Tk|DV)-0.5 + ∆t
(4)
The number of the interrogated data points (n) was optimized based on the signal-to-noise ratio of the data collected. For systems that have low signal-to-noise, higher number of data points is required to minimize false triggers. The higher the signal-to-noise ratio for the system in question, the trigger may be made reliably with lower n. The downside to a high n choice is that the range of ∆t available to optimize the desired Signal Intensity (I) and Peak Resolution (R) combination decreases. To maximize the ∆t range for optimizing S and I, ∆V calculations can be made in the firmware rather than in the software, effectively decreasing the response time of the trigger. Figure 5 shows four electropherograms of runs that use the above computer-controlled handoff scheme. The scheme used the value Th calculated from eq 4. Note that in the four electropherograms in Figure 5 all have comparable Signal intensity, I, and peak separation, S, but have slightly different peak arrival times. The arrival time differences are most likely caused by slight variations in experimental conditions such as gel concentration or buffer conductivity. The fact that the four runs with different peak arrival Analytical Chemistry, Vol. 80, No. 3, February 1, 2008
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times show similar values of I and S indicates that the computercontrolled triggering mechanism is able to control the data quality repeatably even when the operating conditions deviate slightly from ideal conditions. CONCLUSION The system developed for this study has the potential to increase the sensitivity of an immunoassay by 200-fold. Even though the sensitivity increase was demonstrated for a liquidphase homogeneous R-fetoprotein immunoassay, the use of the system developed for this study is not necessarily limited to the model immunoassay. Because ITP works by concentrating the species of interest, the benefits of preconcentration by ITP may be realized in most systems for which appropriate trailing and leading buffers can be found. Moreover, the method of preconcentrating the analyte of interest only available in low concentrations may produce stacking factors that are orders of magnitude greater than the 200-fold demonstrated in this study. Often constraints such as limits on run time, physical current or voltage limits of the hardware, or the assay requirements on the choices of reagents play a more significant role than the theoretical limitation of ITP in determining the maximum stacking factor a system may achieve. Furthermore, the ability to control the signal intensity and peak separations of the species of interest by controlling the delay time
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allows the use of one chip to run a variety of immunoreactions. Many times, the identification of a particular immunocomplex is limited by the ability of the system to distinguish the species of interest from its nearest neighboring species as well as the ability to distinguish the peak of interest from the background noise. The use of delay time to control the gain of intensity and peak separation enables the users to optimize the system often not controllable by any other means. Finally, small changes in the assay conditions such as fluctuations in the buffer concentration or power supply are often unavoidable, when carrying out an immunoassay analysis. It has been shown that for a system that uses an ITP-based preconcentration coupled to a GE-based separation such small fluctuations in the assay condition may lead to large variations in the resulting electropherograms, ultimately leading to inaccuracies in the quantitation of the desired analytes. By using an automatic handoff mechanism that utilizes a feedback control to switch the system from preconcentration to separation, the variability associated with the handoff may be minimized.
Received for review August 13, 2007. Accepted November 1, 2007. AC701709N
