Article pubs.acs.org/ac
Accelerated Surface-Enhanced Raman Spectroscopy (SERS)-Based Immunoassay on a Gold-Plated Membrane Michelle A. Penn, David M. Drake, and Jeremy D. Driskell* Department of Chemistry, Illinois State University, Normal, Illinois 61790, United States S Supporting Information *
ABSTRACT: A rapid and simple SERS-based immunoassay has been developed to overcome diffusion-limited binding kinetics that often impedes rapid analysis in conventional heterogeneous immunoassays. This paper describes the development of an antibody-modified membrane as a flowthrough capture substrate for a nanoparticle-enabled SERS immunoassay to enhance antibody−antigen binding kinetics. A thin layer of gold is plated onto polycarbonate track-etched nanoporous membranes via electroless deposition. Capture antibody is immobilized onto the surface of a gold-plated membrane via thiolate coupling chemistry to serve as a capture substrate. A syringe is then used to actively transport the analyte and extrinsic Raman labels to the capture substrate. The fabrication of the gold-plated membrane is thoroughly investigated and established as a viable capture substrate for a SERS-based immunoassay in the absence of sample/SERS label flow. A syringe pump is used to systematically investigate the effect of flow rate on antibody−antigen binding kinetics and demonstrate that active transport to the capture membrane surface expedites antibody−antigen binding. Mouse IgG and goat anti-mouse IgG are selected as a model antigen−antibody system to establish proof of principle. It is demonstrated that the assay for mouse IgG is reduced from 24 h to 10 min and a 10-fold improvement in detection limit is achieved with the flow assay developed herein relative to the passive, i.e., no flow, assay. Moreover, mouse serum is directly analyzed and IgG level is determined using the flow assay. advantages of SERS-based assays and advances the field of diagnostics; however, efforts that address the time and labor challenges and move immunoassays from the laboratory to the field without sacrificing accuracy are needed to significantly impact disease surveillance and outbreak prevention efforts. The speed at which an immunoassay can be performed is limited by the rate of antigen delivery to the immobilized antibody for binding to occur.33−35 Typically heterogeneous assays rely on diffusional transport, which often leads to long incubation times for large biological molecules with small diffusion coefficients, particularly at low antigen concentrations.36 This diffusion limitation can be even more severe for NP-enabled assays, because of the exceedingly small diffusion coefficient for SERS tags, which leads to even longer assay times, compared to conventional enzyme-labeled antibodies as antigen tags. This is evidenced by most SERS assays which report 2−12 h incubations for each step, e.g., antigen binding and labeling.13,21,22,31,32 Assays requiring several hours are not feasible for POC and field applications, and efforts to shorten SERS assay times are necessary to realize the full potential of
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mmunoassays are essential tools in areas ranging from basic research in life sciences to clinical diagnostics. In addition to the availability and low cost, the accuracy of immunologicalbased methods imparted by the antibody specificity is unparalleled and is the primary reason that these assays are a mainstay in the diagnostics arena. Significant efforts are underway to improve the analytical performance of current immunoassays, with the majority of these efforts aimed at lowering the detection limit using novel detection modalities, e.g., nanowires,1−3 surface plasmon resonance,4−7 quantum dots, 8−11 and surface-enhanced Raman spectroscopy (SERS).12−18 Of these techniques, SERS offers many potential advantages including simultaneous detection of multiple antigens, high sensitivity, photostability, and potential for point-of-care (POC) or field testing with the recent availability of portable hand-held Raman spectrometers. Nanoparticle (NP)-enabled SERS immunoassays have been extensively studied for bioanalytical applications and recently have been comprehensively reviewed.19 NP-based assays employ a SERS tag that consists of a metallic NP modified with a Raman reporter molecule and an antibody that is used to indirectly detect antigen. Since the first demonstration of a SERS assay,20 recent studies have focused on increasing SERS tag sensitivity,21−29 affording multiplexed detection,29,30 and establishing feasibility for clinical samples.31,32 Each of these improvements in the SERS platforms demonstrates the © XXXX American Chemical Society
Received: April 16, 2013 Accepted: August 8, 2013
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heterogeneous SERS immunoassay, we use a previously developed SERS assay scheme13,14,16 and extrinsic Raman labels (ERLs),44 i.e., SERS tag. In addition, a model antibody− antigen system, goat anti-mouse IgG and mouse IgG, was used, although the findings are applicable to other antibody−antigen pairs. A gold-plated polycarbonate membrane filter, as previously developed,45 was modified with antibody to serve as the capture substrate. It should be noted that these goldcoated membranes were previously employed for the SERSbased detection of bacteria.46 However, in that work, the goldplated membranes were not chemically modified and functioned to concentrate the bacteria from water samples based on physical size constraints that prevented flow through the pores; antibody−antigen binding kinetics were not explored or enhanced in that study.46 The focus of this work is the systematic investigation of the effect of sample/SERS tag flow rate on antibody−antigen binding kinetics and the development of an accelerated SERS immunoassay.
SERS detection strategies in immunological-based diagnostic platforms. Several approaches have been investigated to reduce assay time. For example, nanofluidics, which combines antigen confinement and convective flow, has been shown to enhance mass transport and overcome diffusion-limited kinetics.37 Others have utilized electric38 and magnetic29,39,40 fields to actively transport the antigen and label to the immobilized antibody, thereby reducing assay time. Specific to NP-enabled SERS assays, rotation-induced flux41,42 and lateral flow43 platforms have been developed and implemented to reduce assay time. Studies employing rotationally enhanced antigen transport provided fundamental insight into antigen−antibody binding kinetics;42 however, the multistep procedure and bulky platform are not suitable for POC or field testing. SERS lateral flow immunoassays offer the ultimate in simplicity and sufficient speed; however, optical scattering artifacts have been identified that limit quantitative utility.43 Thus, the aim of this work is to develop an immunoassay platform employing SERS tags that reduces assay time and is amenable to on-site analysis, without sacrificing the analytical attributes of SERSbased detection. Here, we immobilize capture antibody on a gold-plated membrane filter and investigate the use of a syringe to flow the antigen and SERS labels through the capture membrane as a means to enhance binding kinetics and accelerate the SERS immunoassay (Figure 1). To establish proof-of-principle for the merits of a capture filter and syringe controlled flow in a
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MATERIALS AND METHODS Reagents. Gold nanoparticles (AuNPs) (30, 40, 50, 60, 80 nm) were purchased from Ted Pella. Sodium borate buffer (50 mM, pH 8.5), phosphate-buffered saline (PBS; 10 mM, pH 7.4), 3,3′-dithiobis[sulfosuccinimidylpropionate] (DTSSP), and goat anti-mouse IgG polyclonal antibody were purchased from Thermo Scientific. Mouse serum was obtained from Santa Cruz Biotechnology. Trifluoroacetic acid and 4-nitrobenzenethiol (NBT) were purchased from Aldrich. Bovine serum albumin (BSA) and purified IgG from mouse serum were acquired from Sigma. Polycarbonate track-etched filters with pore sizes of 0.8 μm (13 mm diameter) and Swinnex filter holders were purchased from Millipore. Oromerse Part B Gold Replenisher was purchased from Technic, Inc. Formaldehyde, tin chloride, methanol, sodium sulfite, nitric acid, sodium chloride, silver nitrate, and ammonium hydroxide all were purchased from Fisher Scientific. ERL Preparation. Extrinsic Raman labels (ERLs) were prepared according to a previously reported procedure.44 Briefly, 1.0 mL 60 nm AuNPs were modified with the addition of 40.0 μL of 50 mM borate buffer (pH 8.5), 2.5 μL of 1 mM DTSSP, and 10.0 μL of 1 mM NBT (in acetonitrile). The suspension was vortexed and incubated for 15 min then centrifuged at 5000g for 5 min. The supernatant was removed and the modified AuNP were resuspended in 1.0 mL of 2 mM borate buffer (pH 8.5). Goat anti-mouse IgG polyclonal antibody (12.5 μL) was added to the suspension and allowed to incubate overnight at 4 °C. Excess antibody was then removed by centrifuging at 5000g, decanting the supernatant, and resuspending the pelleted AuNPs in 1.0 mL of 2 mM borate buffer (pH 8.5) containing 1% BSA for two cycles. After a third cycle, the ERLs were resuspended in 2 mM borate buffer (pH 8.5) with 1% BSA and a physiological equivalent of NaCl (150 mM). The ERLs were then diluted 3-fold (unless specified otherwise) in 2 mM borate buffer (pH 8.5) with 1% BSA and 150 mM NaCl. Dynamic light scattering (DLS) was used to confirm conjugation of the antibody to the AuNP, measured as an increase of ∼20 nm in hydrodynamic diameter,47 and to validate no measurable aggregation of the AuNP resulting from modification (see Figure S1 in the Supporting Information). Gold-Coated Membrane Fabrication. Electroless gold plating of PCTE membranes was performed using a previously established method.45,46 The membranes were first immersed for 3 min in a Sn2+ sensitizing solution containing 26 mM
Figure 1. Schematic and corresponding photographs detailing antigen capture and ERL labeling steps in a two-step assay using a gold-plated membrane as the capture substrate and a syringe for active transport. B
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placed on an XYZ precision stage, and all spectra were collected with an integration time of 5 s. The SERS intensity was collected from five random locations across each membrane. Dynamic Light Scattering (DLS.). DLS was used to measure the hydrodynamic radius of the ERLs prior to use to confirm conjugation of the antibodies and ensure a monodisperse, nonaggregated suspension.47,48 The instrument employed was a BI-90 Plus (Brookhaven Instrument Corporation, New York) equipped with a 658-nm laser and an avalanche photodiode detector (Perkin). Each ERL preparation was analyzed in triplicate with three 1-min runs, using MAS OPTION particle sizing software. Scanning Electron Microscopy (SEM). An FEI-Quanta 450 scanning electron microscopy (SEM) system was used to image the PCTE membranes before and after gold deposition. The sample chamber was maintained under high vacuum and the electron beam was operated at 20 kV. The working distance was in the range of 9−10 mm, and secondary electrons were collected using an Everhardt Thornley detector to generate the image.
SnCl2 and 700 mM trifluoroacetic acid prepared in 50:50 methanol:water. The filters were rinsed three times in methanol (∼30 mL) and then soaked in 29 mM AgNO3 solution with ∼10 drops of ammonium hydroxide for 2 min. After this Ag deposition, the membranes were rinsed twice with methanol and once with water. The silver was galvanically displaced by immersing the membrane in a gold plating solution consisting of 40× diluted Oromerse Part B, 0.127 M Na2SO3, and 0.625 M formaldehyde. The gold plating reaction was carefully controlled at 4 °C for 6 h, then the membranes were soaked in 25% HNO3 for ∼16 h, followed by a final water rinse. Prior to use, the filters were allowed to dry under ambient conditions and stored similarly. Capture Membrane Preparation. A previously described procedure for the preparation of the capture substrate was used with slight modifications.13,14,16 To the gold-plated filters, 150 μL of 1 mM DTSSP was added and allowed to incubate for 1 h at room temperature. The DTSSP-modified membrane was then rinsed with water. Goat anti-mouse IgG polyclonal antibody (150 μL, 10 μg/mL) prepared in borate buffer was then added to the membrane and incubated overnight in a humidity chamber to minimize evaporation. Next, the membranes were rinsed twice with borate buffer and then soaked in 1% BSA prepared in borate buffer for 1 h to block nonspecific binding sites. Following a final rinse with borate buffer, the capture membranes were loaded into filter holders, and 200 μL of 2 mM borate buffer with 1% NaCl was added to the top of the holders to maintain hydration until further use in an assay. Immunoassay Protocol. Mouse IgG calibration standards were prepared via dilution of stock 1.0 mg/mL mouse IgG in PBS. A mouse serum sample was also prepared for analysis by diluting the serum 1:25000 in PBS. For a standard two-step immunoassay, a syringe pump (Pump 11 Elite; Harvard Apparatus) was used to pass sample solution through the capture membrane at a controlled flow rate. The membrane was then rinsed by flowing 0.2 mL of 10 mM PBS through the capture membrane at 1.0 mL/min. Captured analyte was labeled by passing 1.0 mL of ERLs through the membrane at the same rate as the sample. To remove excess ERLs from the bed volume of the membrane, 5.0 mL of 2 mM borate buffer with 1% NaCl and 1.0 mL of water was passed through the membrane at a flow rate of 1.0 mL/min. A syringe was used to pass air through the membrane to expel excess liquid; the membrane was removed from the holder and allowed to dry under ambient conditions prior to SERS analysis. The capture membrane was placed in a 24-well plate to conduct passive assays, i.e., no flow, and incubated with the same volume of sample and ERL labeling solutions as used in the flow experiments. The rinsing procedures for the passive assay were identical to the flow assay to eliminate any effects of flow on bound analyte and ERL which would potentially complicate direct comparison of active and passive transport on antibody−antigen binding kinetics. Instrumentation. Raman Spectroscopy. All SERS measurements were performed on an Enwave Optronics, Inc. ProRaman-L-785B Analyzer, using a diode laser fixed at 785 nm. The laser was focused to a 100-μm spot onto the membrane surface with a 10× objective and the power was adjusted to 30 mW. The scattered light was collected by the same objective and directed into a spectrometer that provided 6.5 cm−1 resolution and was detected by using a high-sensitivity CCD thermoelectrically cooled to −60 °C. Membranes were
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RESULTS AND DISCUSSION Fabrication and Characterization of Gold-Plated Membranes. Gold-plated polycarbonate track etched (PCTE) membranes serve as the capture substrates in the SERS-based immunoassay illustrated in Figure 1. Numerous filters are commercially available, but PCTE membranes offer many advantages, including a narrow distribution of pore sizes, small depth to ensure that the ERLs are accessible by the laser, and durability to withstand necessary handling. However, PCTE membranes exhibit a large fluorescent signal when irradiated with a 785-nm laser, which inhibits detection of SERS signal, and are low protein binding, which prevents direct absorption of capture antibodies onto the surface. A thin layer of gold is plated on the PCTE membrane to circumvent these limitations and enable SERS-based detection strategies. The function of the gold layer is multifactorial. First, a thin gold layer shields the PCTE from laser excitation and eliminates background fluorescence from the membrane. Second, capture antibodies are readily immobilized onto gold surfaces utilizing well-characterized thiol-based cross-linkers. Third, the gold layer provides optimal plasmonics in which the ERL surface plasmon couples to the gold layer surface polariton, generating a large electromagnectic field in the ERL-membrane gap to yield large SERS enhancement factors.49−52 Gold was plated on the PTCE membranes through a method developed by Mennon and Martin.45 Briefly, following a Sn2+ sensitizing step, and Ag+ activation, gold was deposited via electroless deposition. Previous reports have demonstrated that deposition of the gold layer is time dependent, with longer reaction times yielding thicker gold layers.53 Ideally, the PCTE membranes should be fully coated with gold to effectively eliminate background fluorescence while minimizing the reduction in pore size. In an effort to optimize the gold plating step, Ag+ activated membranes with a nominal pore size of 800 nm were immersed in Au plating solution for varying amounts of time, and the background fluorescence of the PCTE was monitored to assess the extent of the gold plating. The background fluorescence signals from the untreated PCTE membrane and membranes prepared with varying Au plating times are provided as Supporting Information (Figure S2). A large fluorescence signal is observed from the untreated PCTE membrane and is significantly attenuated as the gold deposition C
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Antigen-modified membranes were incubated with 1.0 mL of ERLs for 12 h under stagnant conditions, i.e., without flow. The average SERS spectrum for this sample is presented in Figure 3A. This spectrum is characteristic of NBT, with the dominant
time increases. After a 6-h gold plating step the background fluorescence is effectively eliminated, suggesting that a continuous thin gold film on the PCTE membrane is formed (see Figure S2 in the Supporting Information). A photograph of the PCTE membrane before and after the 6-h gold deposition step is provided in Figure 2A. Upon visual
Figure 2. (A) Photograph of membrane filter with and without gold coating. SEM micrographs (20 000× magnification) of (B) uncoated membranes and (C) gold-plated membranes. The scale bar in the SEM images represents 1 μm.
inspection, the gold coating is evident and appears uniform. SEM images were collected from three independent preparations to assess the integrity of the pores and determine the thickness of the deposited gold layer. Figure 2B shows the original pore size of ∼800 nm prior to gold plating. Figure 2C reveals that the pore size is reduced to 566 ± 17 nm, indicating that the gold layer is ∼120 nm thick. Furthermore, the SEM images do not indicate that any pores are clogged as a result of the gold deposition procedure. To confirm pore integrity after gold deposition, 60-nm AuNPs were flowed through filters, and UV/vis spectrophotometry was used to monitor the particle concentration before and after filtration. Absorbance measurements indicate no detectable loss in AuNP concentration (see Figure S3 in the Supporting Information). Effect of Flow Rate on Antibody−Antigen Binding. Initial experiments were designed to establish (1) the ability of the Au-plated membrane to function as a capture substrate in the SERS assay, (2) diffusion-limited kinetics in the absence of active transport, and (3) the feasibility of flow as a viable means to overcome this mass-transport limitation. Toward this end, the target antigen, i.e., mouse IgG, was directly immobilized onto the gold-plated membrane via DTSSP and ERLs were modified with goat anti-mouse IgG antibody and NBT. In this configuration, ERLs bind directly to the antigen-modified membrane in a one-step assay (Figure S4 in the Supporting Information), simulating the second step of the two-step assay illustrated in Figure 1. This design simplifies the two-step procedure while allowing feasibility to be established as noted above and provides a more direct investigation into the effect of flow rate on antibody−antigen binding by eliminating any potential complications from the antigen capture step in a twostep assay.
Figure 3. Effect of flow rate on ERL binding to antigen-modified gold filter: (A) SERS spectra, as a function of flow rate, and (B) intensity of 1334 cm−1 band, as a function of flow rate.
peak at 1334 cm−1 resulting from the symmetric nitro stretch. The intense signal confirms binding of the ERLs to the antigen immobilized on the membrane and establishes that the goldplated membranes can serve as the capture substrate for a SERS-based assay. No signal is detected from a control sample that consisted of a BSA-modified membrane exposed to the ERLs and verifies the specificity of ERL binding to the antigen on the membrane. To highlight the importance of incubation time, antigen-modified membranes were allowed to react with ERLs for 5 min. The SERS spectrum for this short incubation sample consists of the same characteristic NBT vibrational bands as the 12-h sample; however, the intensity of the spectrum is significantly reduced as a result of the shorter incubation time (Figure 3A). These results confirm diffusionlimited binding kinetics and are consistent with previously reported findings.41 The effect of solution flow through the membrane on antibody−antigen binding was investigated as a means to overcome diffusion-limited mass transport and to reduce assay times. A syringe pump was used to systematically control the flow of ERLs (1.0 mL) through the antigen-modified membrane with flow rates ranging from 0.1 mL/min to 6.0 D
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Flow through the membrane may also have implications for the location at which ERLs bind, e.g., membrane surface versus pore walls. The membranes were passively incubated with DTSSP and antigen; thus, antigen is expected to be evenly distributed among the membrane surface and interior walls of the pores. However, theoretical models suggest that, under flowing conditions, ERLs may bind preferentially to the pore walls rather than the membrane surface.54 Surface-bound ERLs are readily interrogated by the excitation laser, whereas only a small fraction of those ERLs bound in the pores are likely irradiated to produce a signal. Thus, it is important to determine where the ERLs bind on the membrane as they pass through the antigen-modified membrane as it will have significant consequences on the measured signal. While SEM imaging of the membranes before and after flowing ERLs through it proved challenging in visualizing ERL binding due to the surface roughness of the gold-plated PCTE membrane, SERS analysis of the top and bottom surface of the membranes does provide some insight into the location of ERL binding. If the majority of the ERLs bind to the interior of the pores rather than the surface of the membrane, then the SERS signal is expected to be similar when analyzing the top or bottom of the membrane. However, if a significant amount of ERLs bind to the surface of the membrane then the SERS signal is expected to be much more intense when analyzing the top surface compared to the bottom surface of the membrane. SERS signal was collected from the top and bottom surface of an antigenmodified membrane passively treated with ERLs (see Figure S5 in the Supporting Information). Under passive conditions, no preferential binding to the pore walls is anticipated, and, as expected, the SERS signal is significantly more intense from the top of the membrane than the bottom of the membrane. Similarly, ERLs were passed through an antigen-modified membrane at 0.2 mL/min and the top and bottom of the membrane were analyzed via SERS (Figure S5 in the Supporting Information). The results show that, like passive incubation, significantly more signal is obtained from the top of the membrane. This suggests that a significant amount of the ERLs are binding to the surface of the membrane. While these results may seem to contradict the theoretical models developed for filtration efficiency of PCTE membranes, it is important to note that the models assume uniform distribution of the pores across the surface and perpendicular orientation of the pores relative to the surface.54 These assumptions are not valid for our membranes which likely lead to more turbulent flow at the surface to facilitate greater than expected interaction with the membrane surface. Nonetheless, these data support that significant binding occurs at the membrane surface under flowing conditions. Effect of ERL Concentration and Volume. The effects of ERL concentration and volume on SERS intensity were investigated to determine if the amount of binding, e.g., ERLantigen complex, was concentration- or mass-dependent. As described above, gold-plated membrane filters were modified with mouse IgG. Anti-mouse IgG ERLs were prepared at three concentrations: 1x (2.6 × 109 AuNP/mL), (1/3)x (8.7 × 108 AuNP/mL), and (1/9)x (2.9 × 108 AuNP/mL). One milliliter of each of the ERL suspensions was then passed through separate antigen-modified membranes at rates of 0.2 and 0.5 mL/min. SERS spectra were collected from each membrane and the intensity at 1334 cm−1 is plotted for each ERL concentration in Figure 5. As expected, the intensity increases as the ERL concentration increases, indicating more bound
mL/min. The experiment was performed in duplicate with each flow rate tested using two independent membranes. ERL binding is quantified as the intensity of the SERS band centered at 1334 cm−1 and the average signal (N = 10, 5 measurements × 2 membranes) is plotted as a function of flow rate in Figure 3B. ERL binding decreases as the flow rate increases. Interestingly, the slowest flow rates of 0.1 and 0.2 mL/min result in a greater signal than the 12 h stagnant assay, suggesting that equilibrium is not achieved in 12 h under passive conditions. Importantly, the results indicate that flow through the membrane can successfully expedite antibody−antigen binding. The fact that binding decreases as flow rate increases suggests that ERLs are actively transported to the antigen on the membrane at a rate that exceeds antibody−antigen binding kinetics and the diffusion limitation is overcome. This is expected, as it has previously been demonstrated that, at sufficiently high rates of active transport, antibody−antigen binding is reaction-limited rather than diffusion-limited.42 However, this trend could alternatively be explained by removal of specifically bound ERLs under flowing conditions due to shear forces to result in decreasing signal with increasing flow rate. Thus, tests were performed to determine the effect of solution flow, and associated force, on bound ERLs. Four membranes were modified with mouse IgG and passively treated with anti-mouse IgG ERLs under identical conditions. Presumably, equivalent amounts of ERLs are bound to each membrane. Each membrane was then rinsed with 2 mM borate buffer using a different flow rate and/or volume of rinse buffer. The results are presented in Figure 4, and equivalent SERS
Figure 4. Average SERS intensity of the 1334 cm−1 band for membranes modified with mouse IgG (positive control), passively incubated with ERLs, and rinsed with borate buffer using different flow rates and volumes. The error bars represent the standard deviation.
intensities are measured for each membrane. The results indicate that flow rates as great as 6 mL/min and rinse buffer volumes as great as 15 mL have no effect on ERL binding and shear forces are not responsible for the decrease in specific ERL binding at higher flow rates. Thus, it is concluded that active transport via flow through the membranes overcomes diffusionlimited kinetics and reaction-limited kinetics are responsible for the trend observed in Figure 3. E
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both control assays in Figure 6. As is evident, the SERS intensities are significantly attenuated for the assay performed
Figure 5. Average SERS intensity of the 1334 cm−1 band collected from membranes modified with mouse IgG and treated with three different ERL volumes and concentrations. Two different flow rates (0.2 and 0.5 mL/min) were employed to establish that the effect of ERL volume and concentration on binding efficiency is conserved.
Figure 6. Calibration curves for mouse IgG using a two-step sandwich immunoassay. Calibration curves were established for two active assays using two flow rates and two passive assays (without flow) employing two incubation times. Data are best fit to eq 1. Dashed horizontal lines represent the minimum detectable signal, defined as the blank signal plus three times its standard deviation.
ERLs. Moreover, the intensity is greater for the slower flow rate, consistent with the data in Figure 3. Specifically, the intensity of the (1/3)x and (1/9)x ERL suspensions are, respectively, approximately 3-fold and 9-fold less than the membrane treated with the 1x ERL suspension. In a subsequent experiment, 1.0 mL of 1x, 3.0 mL of (1/3)x, and 9.0 mL of (1/9)x ERL suspensions were passed through separate antigen-modified membranes at rates of 0.2 and 0.5 mL/min. These parameters were selected such that each membrane was treated with the same total number of ERLs, while the ERL concentrations and volumes varied. Figure 5 shows that the SERS intensity collected from each of these filters (at a given flow rate) are statistically equivalent. It is then concluded that, under these experimental conditions, the amount of antibody−antigen complexes formed is based on the absolute number of antigens or ERLs, rather than the antigen or ERL concentrations (i.e., detects mass rather than concentration). This finding has significant implications regarding assay design. For example, these data suggest that, if available, large volumes of dilute sample can be passed through the membrane to extract a sufficient number of analyte molecules to allow detection. In addition, it suggests that a small volume of highly concentrated ERLs can be used for labeling to further reduce assay time or potentially enhance sensitivity. Two-Step Assay for Mouse IgG. A two-step immunoassay for mouse IgG was performed as illustrated in Figure 1. Gold membranes were modified with goat anti-mouse IgG antibody. Sample solutions were prepared via serial dilution of purified mouse IgG in PBS and ERLs were constructed from 60-nm AuNPs with anti-mouse IgG antibody. ERLs prepared with AuNPs 30−80 nm in diameter were tested, and it was determined that 60-nm ERLs provide the best sensitivity (see Figure S6 in the Supporting Information). Two passive assays, i.e., without flow, were performed as control studies to highlight the significance of active transport. In the first control assay, modified membranes were incubated with the 1.0 mL sample for 12 h, followed by a 12-h incubation in an ERL suspension. The second control assay allowed 5 min incubations in the sample and labeling solutions. The intensity of the NBT band at 1334 cm−1 is plotted as a function IgG concentration for
with 5 min incubations; and thus, provides further evidence for mass-transfer limitations. The anticipated dose−response curve is observed for both assays and each is best-fit to a single-site ligand binding isotherm (eq 1) to establish calibration curves, as previously reported.31 I(Ci) =
Bmax Ci + Ns Kd + Ci
(1)
In eq 1, I(Ci) is the SERS intensity at IgG concentration Ci, Bmax the apparent binding capacity, Kd the apparent dissociation constant, and Ns the SERS intensity resulting from nonspecific binding. The detection limits are better defined from a linear fit to the signals obtained at low antigen concentrations (0−100 ng/mL). Detection limits for the passive assays, defined as the antigen concentration that produces a signal equal to the blank signal plus three times the standard deviation of the blank signal, are 6.9 ng/mL and 80 ng/mL for the 12-h and 5-min incubations, respectively (see Table 1). Table 1. Comparison of Assay Time and Detection Limit of Passive and Active Assays type passive passive active active
flow rate (mL/min)
0.2 1.0
time 24 10 10 2
h min min min
limit of detection, LOD (ng/mL) 6.9 80 0.84 13.5
Two flow assays, e.g., active transport, were performed in which the sample and ERL suspension were passed through the modified capture membrane at a flow rate of 0.2 or 1.0 mL/ min. The data for these two assays are plotted in Figure 6 and best-fit to eq 1. The sensitivity of both flow assays is much greater than the 5-min passive assay, and the assay performed at 0.2 mL/min is more sensitive than the one performed at 1.0 mL/min. These data are consistent with the data presented in F
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Figure 3, and provide additional examples in which active transport overcomes the diffusion limitation such that the binding kinetics are dominated by the reaction rate. Detection limits for the flow assays, as described above, are 0.84 and 13.5 ng/mL for the 0.2 and 1.0 mL/min flow assays, respectively (Table 1). Importantly, the flow assay conducted at 0.2 mL/ min was completed in 10 min, 5 min for each step, yet provided similar sensitivity and a better detection limit than the 24-h passive assay. The specificity of the assay was assessed by analyzing a high concentration sample of human IgG. Anti-mouse IgG antibody was immobilized onto gold-plated membranes. Samples of 104 ng/mL mouse IgG, PBS, and 104 ng/mL human IgG were passed through the capture filters at a rate of 1.0 mL/min. In a second step, anti-mouse IgG ERLs were passed through each filter at a rate of 1.0 mL/min. The capture membranes were rinsed and the SERS spectra were acquired. Each sample was analyzed in duplicate on two independent membranes and SERS measurements from both membranes were used to calculate the average signal and standard deviation. The mouse IgG sample served as the positive control and yielded ∼4000 cts/5 s (Figure 7). In comparison, the PBS and human IgG
Figure 8. Multiple calibration curves for mouse IgG using a two-step sandwich assay to assess assay reproducibility. Open circles represent the average signal from five locations on a single sample membrane. The closed circles represent the average of three independent membranes at each concentration for an assay performed at 0.2 mL/min. The red squares represent the average and standard deviation in signal collected from six independent membranes, with each membrane analyzed at five different locations, for an assay performed at 1.0 mL/min. Dashed horizontal lines represent the minimum detectable signal, defined as the blank signal plus three times its standard deviation.
were collected from each membrane for a total of 30 spectra for each analyte concentration. The average signals are plotted in Figure 8, with the standard deviation represented by the error bars. Similar to the slower flow assay, spot-to-spot variability across a membrane is ∼10%−20%, with ∼15%−30% variability among different membranes of the same concentration. It is important to note that different lots of antibodies can significantly affect the assay results. For example, the data presented in Figures 6 and 8 were carried out using identical conditions and only differ in the antibody used to create the capture membrane and ERLs, yet the absolute intensities vary significantly. Importantly, the effect of flow rate is consistently observed and the assay reproducibility is consistent at 15%− 30% for a given antibody lot. Consequently, a new calibration curve must be generated for each lot of antibodies. Analysis of Mouse Serum for IgG. Protein detection in a PBS matrix is not representative of a typical biological sample. Serum is a more realistic sample that is routinely analyzed for proteins, and it contains a high level of concomitant nontargeted proteins. It is important that a more complex background does not diminish the performance of the flowthrough assay by increasing nonspecific binding or blocking the pores of the capture membrane. To evaluate the feasibility of the membrane assay developed herein to analyze more complex biological samples, unpurified mouse serum was analyzed. IgG levels in mouse serum is in the range of 5−13 mg/mL, which is outside of the dynamic range of this SERS assay. Therefore, the mouse serum was diluted 1:25000 in PBS to reduce IgG levels to an appropriate concentration. While the sample was diluted in PBS, it is important to note that it was not purified and the matrix proteins of the sample are still present in the same relative ratios as the undiluted sample. Mouse serum was analyzed in triplicate using three independent anti-mouse IgG capture membranes (0.2 mL/min flow rate), and the IgG concentration was determined to be 11.8 ± 2.4 mg/mL for the undiluted serum sample.
Figure 7. The average SERS intensity of the 1334 cm−1 band collected from membranes modified with goat anti-mouse IgG antibody and used to analyze samples containing 104 ng/mL mouse IgG (positive control), PBS (blank), or 104 ng/mL human IgG (negative control).
samples resulted in
