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Three-Dimensional Flow-Through Protein Platform R. M. L. van Lieshout,† T. van Domburg,† M. Saalmink,† R. Verbeek,† R. Wimberger-Friedl,*,† M. P. van Dieijen-Visser,‡ and C. Punyadeera§,† Philips Research Europe, High Tech Campus 12, 5656 AE, Eindhoven, The Netherlands and Akademisch Ziekenhuis Maastricht, Maastricht, The Netherlands We have developed a new protein microarray (ImmunoFlow Protein Platform, IFPP) that utilizes a porous nitrocellulose (NC) membrane with printed spots of capture probes. The sample is pumped actively through the NC membrane, to enhance binding efficiency and introduce stringency. Compared to protein microarrays assayed with the conventional incubation-shaking method the rate of binding is enhanced on the IFPP by at least a factor of 10, so that the total assay time can be reduced drastically without compromising sensitivity. Similarly, the sensitivity can be improved. We demonstrate the detection of 1 pM of C-reactive protein (CRP) in 70 µL of plasma within a total assay time of 7 min. The small sample and reagent volumes, combined with the speed of the assay, make our IFPP also well-suited for a point-of-care/nearpatient setting. The potential clinical application of the IFPP is demonstrated by validating CRP detection both in human plasma and serum samples against standard clinical laboratory methods. Immunoassays remain the “gold standard” for protein detection and quantification. Since their introduction in the 1950s, antibodies and other affinity reagents have been developed to improve assay specificity and sensitivity.1,2 In traditional heterogeneous immunoassays such as the enzyme-linked immunoassay (ELISA), long incubation times are used to obtain sufficient sensitivity, because the diffusion of the targets toward the surface is often the ratelimiting step. Protein microarrays are one of the most promising technologies for a wide range of biomedical applications.3-7 However, most of the current protein microarrays are fabricated on solid surfaces,5,7-11 and the assays are performed * Author to whom correspondence should be addressed. E-mail: Reinhold.
[email protected]. † Philips Research Europe. ‡ Akademisch Ziekenhuis Maastricht, Maastricht, The Netherlands. § Current address: Tissue Engineering and Microfluidics Group, Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland, Brisbane QLD 4072 Australia. (1) Ishikawa, E.; et al. Clin. Chim. Acta 1989, 185 (3), 223–230. (2) Oellerich, M. J. Clin. Chem. Clin. Biochem. 1984, 22 (12), 895–904. (3) MacBeath, G. Nat. Genet. 2002, 32 (Suppl), 526–532. (4) Templin, M. F.; et al. Trends Biotechnol. 2002, 20 (4), 160–166. (5) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2003, 7 (1), 55–63. (6) Tanaka, H.; Isojima, T.; Hanasaki, M.; Ifuku, Y.; Takeuchi, H.; Kawaguchi, H.; Shiroya, T. Macromol. Rapid Commun. 2008, 29 (15), 1287–1292. (7) MacBeath, G.; Schreiber, S. L. Science 2000, 289 (5485), 1760–1763. (8) Zhu, H.; et al. Science 2001, 293 (5537), 2101–2105. (9) Houseman, B. T.; et al. Nat. Biotechnol. 2002, 20 (3), 270–274. (10) Kim, D.-N.; Lee, W.; Koh, W.-G. J. Chem. Technol. Biotechnol. 2009, 84 (2), 279–284. 10.1021/ac801244d CCC: $40.75 2009 American Chemical Society Published on Web 05/27/2009
by shaking during incubation. This reduces the reaction kinetics due to slow diffusion of target molecules toward the surface, where capture antibodies are immobilized,12-14 resulting in slow binding kinetics and compromised assay sensitivity and dynamic range. Thus, there is a demand for simple, robust assay devices that are capable of providing more rapidsand more sensitive and quantitativesresults, particularly in a clinical setting. Because the inherent rate of binding of targets to capture probes is mainly determined by the quality of the antibody and the characteristics of the targets, this cannot be addressed from a device configuration point of view.15,16 The device should be conceived in such a way that the inherent binding rate is not compromised by transport limitations of the reactive species. In addition, the device should be highly sensitive for the detection of bound targets (in fact, the label that is attached to the bound targets). Generally, the signal is proportional to the density of labels present at the capture probe, which, in turn, is proportional to the density of captured target molecules. In the further discussion, we use the terms capture probe for the immobilized antibody at the surface and target for the antigen, the presence of which is to be detected. The speed and sensitivity of an assay are inter-related. This can be easily understood from a basic reaction kinetic analysis. Because we are interested in a fast and sensitive detection, we limit ourselves to the initial situation, where only a small fraction of capture probes has reacted with targets; moreover, because we are using a high-affinity antibody, the rate of dissociation is low and can be disregarded in that regime. In fact, the number of bound molecules will scale linearly with the concentration of targets in solution and increase linearly with time: Xt ) konc0t
(
with Xt )
Γt Γ0
)
(1)
where kon denotes the association rate, c0 is the target concentration, and X represents the fraction of reacted capture probes. Γ denotes the concentration of capture probes. With a certain lower detection limit of the sensor for a density of bound labels (Γlim), the time that is required get a significant signal (tlim) will scale inversely proportional to the target concentration: (11) (12) (13) (14) (15) (16)
Nijdam, A. J. J. Proteome Res. 2009, 8 (3), 1247–1254. Karlsson, R. Anal. Biochem. 1994, 221 (1), 142–151. Stenberg, M.; Nygren, H. J. Immunol. Methods 1988, 113 (1), 3–15. Stenberg, M.; et al. J. Immunol. Methods 1988, 112 (1), 23–29. Lin, C. H.; et al. Anal. Biochem. 2009, 385 (2), 224–228. Kusterbeck, A. W. B.; Diane, A. Opt. Biosensors 2008, 243–285.
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Γlim ) Γ0konc0tlim f tlim ∝
1 konc0 Γ0
(2)
To increase the sensitivity without sacrificing speed, one must either introduce an amplification step or a concentration step of the target or of the capture probes. Here, we introduce a technology that is based on an apparent concentration of the capture probes by employing nanoporous substrates. To take full advantage of a high concentration of capture probes on a porous substrate, it is mandatory to avoid local depletion of targets, which is very likely to occur, because of the high concentration of capture probes. Whether or not local depletion occurs is a matter of mass balance between the consumption of targets at the surface and the rate of supply of targets by diffusion and convection. The latter is very much dependent on the flow conditions during incubation. In the absence of flow, the reaction can become diffusion-controlled, which leads to a (Dt)1/2 dependence, where D denotes the diffusion coefficient of the target molecule in solution.13 Because both the consumption and the supply scale linearly with concentration in the short-time and low-concentration regime that is of interest to us, local depletion is governed by the concentration of capture probes. Here, we introduce a way to increase the specific surface area and, at the same time, enhance convective transport by using a microporous or nanoporous substrate in a flow-through arrangement. In this way, we can increase the apparent density of capture probes (and, thus, signal intensity) without suffering from a local depletion of targets. The flow-through concept has an additional benefit of providing stringency, i.e., the strong flow fields (shear stresses) remove weakly bound molecules from the surface, so that nonspecific binding is reduced. As illustrated in Figure 1, a porous NC membrane17 is placed in the flow cell. The sample with the targets and the reagents are pumped up and down through the NC membrane during incubation. The fluorescence signals from the inkjet-printed capture probe spots with diameters of ∼200 µm are determined with the aid of an arrangement as sketched in Figure 1B. We use a fourlight-emitting-diode (4-LED)-based dark-field illumination technique to detect the luminescence after appropriate band-pass filtering with a CCD camera to perform digital image analysis. We present data to demonstrate the proof-of-concept for this ImmunoFlow Protein Platform (IFPP) system. To demonstrate the clinical suitability of IFPP, a sandwich assay was performed to measure the concentration of C-Reactive Protein (CRP) in human serum samples. We chose CRP as a model system to test the IFPP performance because CRP is a widely used inflammation marker. As demonstrated in our present work, the IFPP has the potential to be used for clinical diagnostics. MATERIALS AND METHODS Chemicals and Buffers. All reagents used are of analytical quality; in addition, for all buffers and aqueous solutions, ultrapure water (Milli-Q Synthesis A10, Milli-Q, Bedford, MA) was used. The print buffer PBS was purchased from Sigma-Aldrich (Steinheim, Germany), and other required buffer solutions were made as described by the manufacturer. The detergent Tween 20 and BSA (IgG- and protease-free) were purchased from Sigma-Aldrich (Steinheim, Germany). (17) Pinon, J. M.; et al. Electrophoresis 1990, 11 (1), 41–45.
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Figure 1. (A) Schematic of the ImmunoFlow Protein Platform (IFPP). The plastic rings that contain both the flexible foil (yellow in color) and the antibody-printed nitrocellulose membrane (NC) are clamped together in the rings before being placed into the IFPP. When a vacuum is applied on the IFPP system, the flexible foil will deform, pushing the samples through the NC membranes. (B) Optical setup used to measure the intensity of the spots.
Antibodies and Proteins. The capture probes are ink-jet printed in triplicate. The array experiments were always performed in duplicate, unless otherwise specified. To detect the functionality of the capture probes, a rabbit antimouse IgG and a fluorescent labeled mouse IgG was used. To detect CRP, a pair of mouse monoclonal anti-CRPs (capture (clone 4) and detection (biotinylated clone 2)) was used (Biotrend Chemikalien GmbH, Ko¨ln, Germany). Recombinant CRP was purchased from Biotrend Chemikalien GmbH (Ko¨ln, Germany). Spots of fluorescently labeled (Alexa 633) donkey antisheep (Invitrogen, Carlsbad, CA) were used as an internal calibrator. Fluorescently labeled strepavidin (Alexa 633) was purchased from Invitrogen (Carlsbad, CA). Microarray Substrates. We used a Protran BA 85 NC membrane (with a thickness of 150 µm and a mean pore size of 0.45 µm) from S&S Whatman GmbH (Dassel, Germany) as the substrate. The protein binding capacity of the NC membrane is 80 µg/cm2, according to the manufacturer. Inkjet Printing of Antibodies. Prior to printing, capture probes were dissolved in PBS at pH 7.4. Capture probes are deposited onto NC membranes using inkjet print technology that was developed at Philips Research. A dedicated piezo-driven single nozzle is used to inkjet-print capture probes onto NC membranes. The applied nozzle is a micropipet with a 50-µm-diameter opening with which droplets with a volume of 100-150 pL can be generated at a frequency of 100 Hz. Twenty-seven droplets were dispensed at each spot, resulting in a spot diameter of 180-200 µm. The pitch of the spots in the array was set at 400 µm. Printing was conducted in a clean room (Class 10000) at 60% relative humidity. An automatic nozzle cleaning procedure is implemented to prevent cross-contamination between different print fluids. We inkjet-printed the mouse-anti-CRP (8 µM) capture probes in triplicate, to account for variations caused by the substrate inhomogeneity. Capture probe spots of AF633 donkey-antisheep solution (1 µM) were used as an internal calibrator to normalize signal intensity between different arrays. After inkjet-printing the capture probes, the NC membranes were blocked by incubating in PBS buffer with 5% BSA for 1 h at room temperature (for all experiments).
Recovery of Printed Antibodies on NC Membranes. Before and after blocking and washing, an image was taken and the intensity of the capture probe spots was measured. The recovery of the printed capture probes was calculated as the ratio of luminescence intensity after and before. All intensities are normalized for the excitation intensity and integration time. The average intensity of the areas outside the spots was subtracted as background intensity from the averaged intensity measured at the spot. Binding Kinetics: CRP Sandwich Assay on “No Flow” and “Flow-Through” IFPPs. We used a sandwich CRP immunoassay with a biotinylated detection antibodies. Two incubation methods were deployed: a no-flow method and a flow-through method using the IFPP. The no-flow method uses printed NC membranes that are cut and placed in a 24-well culture plate. Five hundred microliters of CRP target solution is added and incubated at room temperature. After the NC membranes are washed three times (using PBS + 0.05% Tween-20, 500 µL), they are incubated with a biotin-labeled detection antibody (87.7 nM) for 1 h at room temperature. This is followed by another washing step, and 500 µL of AF 633-labeled streptavidin (8.8 nM) is added and incubated for 30 min at room temperature. The signal is measured after washing. In the flow-through method on the IFPP, 70 µL of CRP is pumped through the NC membrane for 5 min at room temperature, with a silicone membrane actuated by a vacuum pump, the pressure of whch is set at 475 mbar. The sample fluid is pipetted off and 70 µL of biotinylated anti-CRP (7.7 nM) is added and pumped for 5 min at room temperature (similar settings as those previously described). After pipetting the Ab solution out, 70 µL of AF633 labeled streptavidin (8.8 nM) is pumped through the membrane for 1 min. The total assay time is 11 min. After the last incubation step, the membranes were air-dried and imaged. Storage of Printed NC Membranes. Membranes were stored in darkness at 4 °C. We were able to store printed NC membranes without observing a significant signal intensity drop for a maximum of three months. Clinical Validation. To validate the IFPP for its suitability for use as a diagnostic test, 20 clinical serum samples were analyzed. The samples were collected at the Academic Hospital Maastricht, under medical ethical approval and blindfolded. At the routine clinical chemistry laboratory in Maastricht, two different methods were used to determine the concentration of CRP in human blood samples: a highly sensitive CRP method (1-50 mg/L), using the BN Pro-Spec from Dade Behring (Dade Behring, Liederbach, Germany), and, for high CRP concentrations (>50 mg/L), the LX20-hs CRP Synchron LX 20 PRO apparatus was used, which involves a turbidimetric method and was operated on a BD instrument (Beckman Coulter Inc., Fullerton, CA). These methods have previously been tested for their suitability as clinical diagnostic tests.18,19 For the analysis on our IFPP, the samples were diluted with a buffer to a working concentration of 10-500 pM. ELISA for CRP. A CRP ELISA kit from Immuno-biological Laboratories, Inc. (Hamburg, Germany) was used to demonstrate IFPP assay performance and test the functionality of the CRP capture probes that were used on the IFPP. The CRP ELISA is (18) Rothkrantz-Kos, S.; et al. Annu. Clin. Biochem. 2003, 40 (Pt 4), 398–405. (19) Rothkrantz-Kos, S.; et al. Clin. Chem. 2002, 48 (2), 359–362.
briefly described as follows. CRP was added at varying dilutions to mouse antihuman CRP coated polystyrene wells and incubated for 1 h on a shaker at room temperature. After incubation, plates were washed multiple times, to remove unbound targets, and an enzyme-conjugated secondary Ab (HRP-antihuman CRP) was incubated with the CRP and primary Abs for 30 min. After rigorous washing, an enzyme substrate (that contained H2O2 and TMB) was added and incubated for another 30 min, followed by multiple washing steps. The enzymatic reaction was stopped with H2SO4 (which was indicated by the color change blue to yellow) and measured at a wavelength of 450 nm, using FluorStar OPTIMA equipment (BMG Labtech GmbH, Offenburg, Germany). Imaging and Data Analysis. Protein microarrays were imaged using an in-house-built LED-CCD setup that has a lower limit of detection of ∼500 Alexa AF633 fluorophores/µm2 on the NC membrane, thus providing high-quality images of protein microarrays. Image processing was performed with the Labview Software (Austin, TX). After measurement of fluorescence, the signal intensity was corrected by subtracting the background intensity (which is defined as four blank areas outside the specific signal spot). The intensity was normalized against the reference spots (AF 633-labeled antibodies) as follows: I)
100 × Icorr Icorr(ref)
(3)
The lower limit of detection (LOD) was defined as the mean value of the blank plus three times the standard deviation (SD) of the blank: LOD ) (blank) + 3SD RESULTS AND DISCUSSION Uniformity of Printed Capture Probe Spots. The membrane uniformity was evaluated in terms of signal intensity variation across the entire NC membrane, as shown in Figure 2. This was achieved by printing AF 633 labeled donkey-antisheep IgG at a fixed concentration (1 µM), covering the entire NC membrane and leaving only spaces around the corner markers. Figure 2 also shows that the signal intensity on the top of the microarray is less than that on the bottom part. The difference is ∼10%. This was observed with all different types of capture probes that have been printed on NC membranes. Generally, the area just below the center of the NC membrane was determined to have the highest signal intensities. To minimize the intrinsic variations, we inkjet-printed antibodies in the middle part of the NC membrane in all assay experiments. Binding Kinetics on IFPP versus No-Flow. As discussed in the beginning of this paper, our objective is to increase the sensitivity and the speed of immunoassays by increasing the specific capture surface area and, therefore, the number of capture probes. An increase in capture probe density should lead to an increased binding of targets, unless the binding is limited by the diffusion of targets to the capture probes. To address the importance of active replenishment of target molecules, we performed two different assays (IFPP versus no-flow), with identical microarrays. The CRP (c ) 100 pM) incubation time was varied, while biotinylated detection Ab and streptavidin incubation Analytical Chemistry, Vol. 81, No. 13, July 1, 2009
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Figure 2. (A) An image of a microarray (10 × 12 spots) after printing. (B) Intensity linescan of column 4 from the image shown in panel A (outlined in red).
binding isotherms with a linear regime at short times (see the beginning of this paper). In the second case, the increase will be equal to the supply of targets by definition, i.e.,
( )
dΓt As ) c0Φs ) c0 Φ dt A
Figure 3. Comparison of CRP binding kinetics on IFPP (flowthrough)- and “no-flow”-based microarrays, depicting the fluorescence intensity versus incubation time with the target solution (c ) 100 pM). The incubation times for the detection of the Ab and Streptavidin solutions were kept constant. Note: the lines are present only to guide the eye.
times were fixed at 1 h and 30 min, respectively, for both methods. A comparison of fluorescence intensity that results from the IFPP (flow-through) and “no-flow” assays is shown in Figure 3, as a function of CRP target binding time. As can be seen, the slopes of the signal-time curves for “flow-through IFPP” and the “noflow” case differ by a factor of 10. This demonstrates the importance of flow (i.e., the transport of the target molecules to the capture probe sites). In the following we attempt to explain the observed behavior in a qualitative way, in terms of the effects of active pumping, which distinguishes the IFPP. Because of the heterogeneous nature of the NC porous membrane and the free surface flow inside the device, it is not feasible to attempt a quantitative description. Here, we only want to present some scaling consideration that can help to understand the behavior and potentially further improve on the IFPP technology. For a simplified description of the target binding inside the membrane during a single pass of solution (one stroke of the actuating membrane), one can distinguish two extreme cases, i.e., that of ambient assay conditions20 and that of instant depletion of targets. In the first case, the increase of bound targets (and, consequently, the signal) with time would scale following Langmuir 5168
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(4)
where Φ is the volumetric flow rate, A denotes the area of the membrane, and As represents the area of the spot with capture probes. To determine which of the two extreme cases, as described previously, is a better description for the experimental situation inside the porous NC membrane and whether we can understand the results depicted in Figure 3, the system is described as follows. We have a NC membrane with an area A, and a total thickness Tm. The membrane is loaded with a volume V of sample solution containing a target with a concentration c0. On the membrane, we find a number of spots (S) that have an area As and a number of capture probes (N), which is calculated from the number of antibodies printed on the membrane multiplied by the recovery (R) after washing: N ) cpVpNAR
(
with R )
Sw S0
)
(5)
Here, NA denotes Avogadro’s number, cp is the concentration of (Ab) in the print buffer, Vp is the printed volume, and S0 and Sw represent the fluorescence intensity before and after washing, respectively. (We assume a linear relationship between fluorescence intensity and the number of dye labels, which will not hold generally. However, because the observed recoveries are typically >50%, the possible error is not expected to affect the outcome of the scaling considerations.) The solution is pumped up and down through the NC membrane continuously in a cyclic fashion with a cycle time tc. We further assume that the solution is mixed perfectly every time it leaves the NC membrane, so that, in every cycle, the solution that enters the capture area has a concentration equal to the average concentration. To estimate the number of molecules bound (20) Finckh, P.; Berger, H.; Karl, J.; Eichenlaub, U.; Weindel, K.; Hornauer, H.; Lenz, H.; Sluka, P.; Ehrlich-Weinreich, G.; Chu, F.; Ekins, R. In Proceedings of the UK NEQAS Meeting, Vol. 3; 1998; p 155.
during their passage through the NC membrane, we assume ideal conditions of binding with an estimated value for kon. We must determine the fraction of target molecules that is bound during each passage through the NC membrane, as well as the total number of target molecules captured versus the number of target molecules present in the original solution. Because we have only a small area of the NC membrane covered with capture spots, the screened volume per passage (Vs) is only a small fraction of the total volume.
( )
Vs As )S V A
(6)
Under the pertinent experimental conditions, a capture spot is 1 /600th of the total membrane area. Our experiments were performed with three identical spots, which means that 1/200th of the volume is screened every passage. (The cycle time is 3 s, which means ∼1.5 s for a single passage.) If instant depletion would occur inside the NC membrane during every passage, the concentration of the target in the total sample would decrease by 0.5% with every passage, which would mean a reduction to ∼5% of the starting concentration after 300 cycles (900 s) and a complete depletion of the total volume before 1200 cycles (the last point in the graph of Figure 3). The graph in Figure 3 indicates that the signal is still increasing after that many cycles, so clearly the assumption of total depletion inside the membrane does not reflect the experimental results. For a more-realistic estimation, one can derive the consumption of targets from short-term linear response, as expressed in eq 1. The temporary relative depletion of targets during a single passage through the membrane (Ψ) can be derived by relating the consumption to the supply of target by the flow: Ψ)
Nkonct Nkon NX ) ) ΦsNAct ΦsNAct ΦsNA
(7)
In our experiments, the number of capture probes per spot (N) is typically 1 × 1010 (as determined with the aid of eq 5). Assuming a reasonable value for kon of 1 × 105 M-1 s-1 and taking the flow rate as 70/600 µL in 1.5 s, the depletion Ψ will be ∼0.05. This means that 5% of the targets of the screened volume are captured during every single passage of the solution through the membrane. Because the three spots only screen 1/200th of the solution, the decrease in target concentration in the total solution is 0.025% per passage, which means that approximately one-quarter of the targets would be captured after 1200 cycles. This is in good agreement with the graph of Figure 3, although the few experimental points clearly do not allow any more precise statement. One should bear in mind that this figure scales with the kon value of the binding reaction, which we have not determined independently. It is also only valid in the regime where the fraction of capture molecules that have reacted is small. With increasing coverage, the consumption will be reduced gradually (following Langmuir kinetics), until, at equilibrium, net consumption ceases. One must take into account that the equilibrium coverage is determined also by the dissociation reaction constant koff, which we have disregarded in the scaling considerations so far, because we are particularly interested in the short-term response. In the shortterm regime, the relative depletion of targets is independent
of target concentration as long as there is a sufficient number of capture probes available. Under the conditions of our experiments, the number of capture molecules (1 × 1010) is larger than the number of target molecules (4.2 × 109). For higher concentrations of targets, saturation of the capture probes can limit the maximum signal as will become evident from dose response curves shown below. With the estimated consumption of 5% of the targets upon a single passage, the effective target concentration will not vary by more than 5% throughout the NC membrane. Therefore, one can expect to obtain a homogeneous distribution of bound targets through the three-dimensional (3D) spot. The signal increase with time, as depicted in Figure 3, is more or less linear for half an hour. This would mean that the number of targets bound per cycle is constant and, consequently, we still have not attained either saturation of the capture probes in the spot or overall depletion of the solution after half an hour. This is consistent with the estimations made about the number of bound molecules and the overall depletion as derived from eq 7 with the assumptions previously made. With a kon value of 1 × 105 M-1 s-1 after half an hour, ∼1 × 108 targets will be bound, which is only a small fraction of the number of capture molecules (∼1.5 × 1010). This means that the capture surface is not yet saturated. The height of the signal is equivalent to 4.6 × 108 AF 633 labeled Abs, as obtained from a calibration curve with printed AF 633-labeled Abs. This difference can be largely explained by the fact that, in the assay, biotinylated detection antibodies are used, with an average of three biotin molecules per antibody (the degree of labeling (DOL) is 3), as given by the supplier, to which fluorescently labeled streptavidin is then coupled. The DOL of streptavidin is 3, which is similar to the DOL of the fluorescently labeled antibody used for calibration. This means that every captured target carries approximately nine fluorophores, whereas in the corner markers, every antibody carries three fluorophores. Now we want to look at the no-flow condition. As shown in Figure 3, the signal increase is only 1/10 that of the flow-through method. Apparently, the lack of replenishment of targets by active flow leads to a drastic reduction in binding rate. As a matter of fact, the number of capture probes present in the 3D spot is much larger than the number of target molecules inside the same volume at the pertinent concentration. The exact volume of the spot is actually difficult to determine precisely, because the spread of the printing solution in the depth direction of the membrane is unknown. Based on results of confocal microscopy measurements, the spot depth is estimated to be approximately one-third of the membrane thickness (i.e., 50 µm), which results in a volume of ∼1.6 nL, half of which is NC polymer (∼50% porosity). Based on this, the number of capture probes as printed corresponds to a virtual concentration (cv) of 20 µM. (The term “virtual” reflects the fact that the capture probes are not in solution but rather are bound to the porous structure.) Even with only a fraction of capture probes functional, the virtual concentration is much higher than the employed concentration of targets. Without replenishment, depletion is likely to occur at some point, even at high target concentrations. Because of the submicrometer morphology, convection inside the membrane is not possible, so that, in the case of the no-flow method, only diffusion can supply new targets inside the spot. Analytical Chemistry, Vol. 81, No. 13, July 1, 2009
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With an expected, rather slow supply of targets by diffusion, one can expect a coupling between diffusive transport and binding reaction. Because we have no precise values for the diffusion and reaction parameters, we only provide order-of-magnitude estimations here to help us understand the difference between flowthrough and no-flow conditions. If consumption by reaction is faster than supply by diffusion, the targets will not be able to reach the center of the capture spot but will already be bound at the outer regions of the spot. For a first-order estimate, we relate a characteristic depletion time (tx) to a characteristic diffusion length (Ld). The parameter tx is derived from the time that is required to consume the target inside the capture spot. The expression for the depletion of target during a single passage through the membrane can be denoted as Ψ)
Nkonc0t ) cvkontx c0NAVspot
(8a)
from which the characteristic depletion time can be derived from eq 8b ∀: Ψ ) 1
tx )
1 cvkon
(8b)
As one can see, the depletion time is reduced by having a high capture probe density (cv) and a high affinity (kon). The distance to which targets can be replenished by diffusion inside the NC membranesthe so-called penetration depthsscales with the diffusion coefficient according to Fick’s law. By equating penetration time to depletion time, we can arrive at an estimate for the penetration depth of targets, i.e., the depth in the membrane to where we can expect to find bound targets (LD): LD ) √4Dtx )
4D cvkon
(9)
where D is the diffusion coefficient. With D ) 1 × 10-11 m2/s for larger proteins, such as CRP (Mw ) 110 kDa), and kon ) 1 × 105 M-1 s-1, as used previously, LD ≈ 5 µm. This means that we would find bound targets only in an outer shell of a capture spot 5 µm thick and, consequently, only that portion will contribute to the fluorescence signal. The core of the 3D capture spot is not reached by the targets, which is in contrast to the flow-through situation where we expect to have a practically homogeneous distribution of bound targets inside the spot. Despite the fact that this is only an approximate estimation, it explains very well the observed 10-times-slower increase in signal, as compared to the flow-through situation. For a more precise analysis, one would also be required to take into account the decreasing contribution of fluorescent labels with increasing depth inside the membrane, because of possible scattering losses under the pertinent conditions. This demonstrates that, to take advantage of the high density and number of capture probes on a porous substrate, it is necessary to actively pump through the solution. The flow-through principle leads to a 10-times-faster increase in signal, which means 10-times-shorter assay times, as compared to the no-flow situation with a porous membrane. Although we have not compared this to conventional, nonporous, two-dimensional (2D) substrates, it can be expected that the gain will be even much greater, because 5170
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Figure 4. Confocal fluorescence microscopy (CFM) images of the cross sections of capture spots. Top: after CRP assay under the noflow condition ([CRP] ) 1 nM); middle: after CRP assay under flowthrough conditions ([CRP] ) 1 nM); and bottom: inkjet-printed fluorescently labeled capture antibody (donkey-antiSheep-AF633, 1 µM, print volume ) 4.9 nL). Images were taken with Leica Model TCS SP5 (Wetzlar, Germany). To reduce scattering, the membranes were immersed in Immersol 518F (Zeiss, Oberkochen, Germany).
the number of capture probes per projected surface area is much lower than in the case of a membrane layer of 5 µm (LD ) 5 µm). The scaling equations presented here are based on approximations and can only indicate the order-of-magnitude effects. Interestingly, they confirm the experimental findings very well. To further support the results of the simple scaling as presented previously, 3D distributions of fluorescence were determined by confocal fluorescence microscopy (CFM) measurements. Membranes were immersed in an index matching liquid (Immersol 518F, Zeiss, Oberkochen, Germany) with n ) 1.52) after the assay and scanned. The scanning was conducted in planes parallel to the membrane and stepping in the thickness direction. In Figure 4, the resulting fluorescence distributions are depicted in the cross sections of the membranes, which were constructed from the scanned images. The spots were printed from the top side of the pictures shown in Figure 4. As can be seen in the picture at the bottom, the capture molecules apparently do not penetrate the total membrane during printing but remain in the upper 50 µm. The fluorescence is quite homogeneous in the case of the printed labeled Abs. The small variations are caused by the inhomogeneity of the membrane structure itself. In the case of assaying with no-flow conditions (top picture), only the rim of the spot volume shows high fluorescence. The intensity at the bottom of the spot is not visible very well in the image. This can be explained by the fact that, because of the placement in the Petri dish, diffusion from the bottom is hampered. The fluorescence intensity distribution is in very good agreement with the estimated depth of binding, based on eq 9. As predicted the target molecules are bound before they can diffuse to the center of the spot. In the case of the flow-through incubation (middle picture), the fluorescence intensity is much more homogeneous in the thickness direction of the membrane. When comparing the false color images, one must take into consideration that CFM images are not necessarily quantitative, in terms of concentration of fluorophores, and the software automatically scales the images to the highest value. The intensities were not corrected for scattering losses, because an index-matching liquid was used. Binding Kinetics of the Detection Antibody. As an attempt to further reduce the total assay time from 11 min to 7 min on the IFPP, we investigated the binding kinetics of biotinylated detection antibodies by varying the incubation times over a range of 0-10 min for different concentrations of CRP target (1 nM-1 pM). It turns out that the signal intensity is almost saturated already at the first measurement point, i.e., after 1 min. From this
Figure 6. Correlation of the detection of CRP in patient samples (n ) 20) by the flow-through assay and clinical assays, using the Prospec method (for concentrations of 0-50 mg/L) and LX 20 PRO (for concentrations of >50 mg/L).
Figure 5. Dose response curve (DRC) of CRP (A) in a buffer on IFPP (assay time ) 11 min) and “no-flow” method (assay time ) 2.5 h) (B) in 50% bovine serum (IFPP only). The total sample volume is 70 µL. The inter-assay and intra-assay variations are 7.2% and 7.0%, respectively.
experiment, it is clearly possible to reduce the detection antibody incubation time from 5 min to 1 min. The same holds for the binding of streptavidin, which is known for its high affinity. The incubation time could be reduced to 1 min with a reduction in signal intensity of only 20%. Dynamic Ranges and the Lower Limit of Detection on the IFPP. Dose response curves (DRCs) for CRP, obtained with the IFPP under flow-through and with no-flow conditions, are depicted in Figure 5A. As one can see, the flow-through DRC is shifted vertically by one decade, compared to the no-flow situation. The saturation is reached at 1 nM for both cases, which indicates that this is due to the equilibrium of binding between CRP and the antibody that we used. Recall that the assay time in the flowthrough situation is only 11 min, compared to 150 min under the no-flow condition. Because of the higher signal, the flow-through approach has potentially more “space at the bottom”, which means extra dynamic range. Currently, this space is not used, because the blank level is high, as a consequence of the limited specificity of the antibodies and streptavidin under the present assay conditions. Our results demonstrate that a limit of detection (LOD) in the sub-pM range is feasible even at the extremely short assay times and small sample volume employed. Using the IFPP with the LED-CCD setup, we were able to detect 1 pM of CRP also in complex matrices, such as serum, as demonstrated in Figure 5B. In this case, the signal is lower than that in the buffer. This is probably due to a less-efficient binding caused by matrix effects. IFPP Sandwich Assay for the Detection of CRP in Human Clinical Samples. To demonstrate the clinical application of the IFPP, we compared our method with two methods used in a routine
clinical chemistry setting for CRP and hs-CRP, respectively. To cover the entire concentration range, high-concentration samples were diluted before the assay with the IFPP. In Figure 6, the results of the IFPP are compared with the commercial assays. We find a reasonably good correlation between the methods. significant deviation is observed only at the highest concentration of the hs-CRP method (i.e., close to 50 mg/L). One must remember that our assay is not optimized. The comparison still indicates that the IFPP is suitable to be used as a diagnostic device. CONCLUSIONS In summary, we have developed a novel three-dimensional (3D) flow-through protein microarray technology based on a porous substrate and vacuum-driven actuation. We demonstrated the detection of 1 pM of CRP with a total assay time of 7 min and a sample volume of 70 µL. Using simple scaling considerations, we can support the experimentally observed 10-fold increase in signal, compared to no-flow incubation. This new 3D protein microarray is particularly attractive for clinical diagnostics with reduced assay time and small sample and reagent volumes. In addition, IFPP platform also holds potential to being developed into a point-of-care device to be placed either at the intensive care unit/emergency room (ICU/ER) and/or general practitioner’s office. Because of the printing of capture probes on the membrane, it holds potential for high degrees of multiplexing, which make it well-suited as a life science tool for detecting multiple targets simultaneously in a large number of samples. ACKNOWLEDGMENT The authors wish to thank Frank Jaartsveld and Theo Loring (for their support in the mechanical design of the device), Aleksey Kolesnychenko (for his contributions to the optical setup), and Faustin Usabuwera (for printing).
Received for review June 18, 2008. Accepted April 24, 2009. AC801244D
Analytical Chemistry, Vol. 81, No. 13, July 1, 2009
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