Technical Note pubs.acs.org/ac
Lab-on-a-Bubble Surface Enhanced Raman Indirect Immunoassay for Cholera V. L. Schmit,† R. Martoglio,‡ and K. T. Carron*,†,§ †
University of Wyoming, Chemistry Department, 1000 East University Avenue, Laramie, Wyoming 82071, United States Department of Chemistry and Biochemistry, DePauw University, 602 South College Avenue, Greencastle, Indiana 46135, United States
‡
ABSTRACT: We describe a novel sandwich assay based on surface enhanced Raman scattering (SERS) comprised of buoyant silica microspheres coated with antibodies against the β subunit of the cholera toxin (CT), and gold nanoparticles tagged with a Raman reporter, shelled with silica and coated with antibodies against the β subunit of the CT. Together these components couple to form a sandwich which, after incubation, floats on the surface of the sample. The buoyant silica microparticle/nanoparticle reporter combination has been coined a lab on a bubble (LoB). LoB materials may provide a platform for rapid detection of antigen in solution and offers advantages over lateral flow or magnetic pull-down assays. The Raman reporter provides a unique and intense signal to indicate a positive analysis. Our limit of detection for the β subunit of the CT in a buffer based system is 1100 ng.
S
urface enhanced Raman scattering (SERS) assays have been proposed as effective analytical methods due to the robustness of properly prepared nanoparticle materials,1 the large dynamic range of single molecules to high analyte concentrations,2 the selectivity of Raman spectroscopy, and development of small portable Raman devices to read the assays.3 We recently demonstrated an interesting direct SERS assay that employed buoyant silica bubbles derivatized with gold nanoparticles (AuNP).4 It was demonstrated that the buoyancy could pull the AuNP coated silica bubbles, coined lab-on-a-bubble (LoB), from the sample volume to a compact monolayer of LoBs on the surface of the sample. Direct SERS assays have been demonstrated with colloidal AgNP or AuNP, with SERS active substrates, and with AuNP modified paramagnetic particles. Many schemes have been used to enhance the adsorption of analytes to the fairly unreactive noble metal surfaces. The significance of the LoB direct assay concept stems largely from the stability of the nanoparticle coating in contrast to the inherent instability of colloidal particles. Figure 1 illustrates a LoB indirect assay. This assay, rather than utilizing AuNP coated LoBs, has LoBs that are coated with an analyte binding reagent. The analyte contains multiple binding sites such that it can also bind to an AuNP reporter (NPR) coated with analyte binding reagents. The NPR consists of an AuNP core, single or multiple AuNPs, covered with a submonolayer coating of a coupled strong Raman scatterer and a protective shell of SiO2. The NPRs have the advantage of robustness in comparison to a colloidal AuNP. The relative area of the of the silica bubble to the shell nanoparticle is about 4 × 104, making it likely that multiple analyte bindings can occur at a single LoB. © 2012 American Chemical Society
Figure 1. Conceptualization of an indirect LoB assay for cholera. The components (left) consist of a cholera-antibody derivatized silica bubble (LoB), the cholera-antigen (CT-AG), and an antibody derivatized silica shelled AuNP reporter. For this project, the Raman reporter is 1,2-bis(4-pyridyl)ethylene (BPE). The resulting reaction between antigen and the LoB components is illustrated to the right. The relative dimensions are exaggerated to show the AuNP reporters. Multiple reporters/bubbles are possible and were observed by SEM imaging.
We chose cholera as the model system to demonstrate a LoB indirect assay. Vibrio cholerae is the causative agent of cholera, a highly contagious and commonly fatal bacterial infection of the gastrointestinal tract. Death can occur within hours of infection Received: January 24, 2012 Accepted: April 2, 2012 Published: April 2, 2012 4233
dx.doi.org/10.1021/ac300242k | Anal. Chem. 2012, 84, 4233−4236
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Technical Note
if not treated immediately and is usually due to hypovolemic shock or acidosis.5 Individuals infected and actively shedding V. cholerae routinely demonstrate 107 to 108 colony forming units (CFU)/mL of feces. The most common method of identifying cholera in environmental samples is traditional microbiology: enrich samples for infectious agents by growing them on selective media and further selection and identification of a serotype through a series of biochemical tests which take approximately 8 days for a conclusive determination.6 Other tests have been introduced in the search for a quick and effective V. cholerae identification: polymerase chain reaction (PCR) following enrichment steps,5 direct cell duplexing PCR for immediate identification of infectious strains,7 digoxigenin labeling (DIG) or radioactive hybridization of colonies for selection of infectious strains after initial colony growth,6 and various immunoassays of V. cholerae colonies directly imaged by microscopy or Western blotting.8 The U.S. Food and Drug Administration couples bacterial enrichment steps to PCR identification of pathogenic strains.9 A rapid, accurate diagnostic assay for the presence of cholera toxin (CT) in either a water sample or a patient sample would significantly benefit those in outbreak areas.
Figure 2. SEM images of a positive LoB assay. Images A, B, and C are acquired with refelected electrons to enhance the physical structure of the LoB materials. Image D used backscattered electron detection to visualize the captured AuNP particles. Note that many are AuNP combinations.
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MATERIALS AND METHODS LoB Activation and Antibody Attachment. LoBs (3 M S60 glass bubbles) were activated with 10 N sulfuric acid overnight. Bubbles were silanized with 1:10 3-aminopropyltriethoxysilane (APTES) in methanol overnight and washed extensively in methanol (MeOH). Bubbles were resuspended in 3 mL of HPLC grade water. Following APTES silanization, antibodies were activated with the carbodiimide 1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride (EDC). In total, 1 μg of CT subunit B antibody (anti CT antibody) (Abcam 34992) was added to the reaction with EDC and activated and silanized LoB solution. Preparing and Shelling Au Nanoparticles. Gold nanoparticles were prepared using the citrate reduction method described by Frens in 1973.10 Colloids were sized using SEM and were an average of 50 nm in diameter. Nanoparticle concentration was determined as described by Haiss et. al.11 After UV−vis spectroscopy and the calculations from that work, we determined the concentration of our nanoparticles to be 6.02 × 1010 nanoparticles per mL. A volume of 4 mL of fresh colloids were labeled with 50 nM 1,2-bis(2-pyridyl) ethylene (BPE) and added to 20 mL of isopropanol (99%) at room temperature while stirring. Colloids were shelled with silica as detailed in Lu et al.12,13 The SEM image in Figure 2D shows that many of the NPR are paired AuNPs. This is significant as it has been demonstrated that paired AuNPs provide larger enhancements.14 LoB Immunoassay. Antibody conjugated LoBs were blocked with nonfat dry milk in PBS and incubated for 10 min shaking at room temperature prior to addition to reaction. Shelled, tagged colloids were incubated with a 1:500 dilution in PBS anti CT antibody (original concentration 1 mg/mL) and incubated for 20 min shaking at room temperature to allow antibodies to adsorb to the silica surface. Following antibody adsorption, colloids were blocked with nonfat dry milk in PBS and incubated for 10 min shaking at room temperature prior to adding the colloid component to reaction. Recombinant β subunit CT (concentration, 1 mg/mL) (Sigma Aldrich C9903) was added at varying concentrations to each reaction. The standard addition experiment antigen addition description is as
follows: (1) unknown concentration of CT (final volume in this reaction is 50 μL), (2) unknown + 2500 ng of CT, (3) unknown + 5000 ng of CT. Antibodies were attached to LoBs in Eppendorf low binding tubes (catalog no. 0030 108.116) using EDC. Prior to each assay, antibodies were adsorbed to shelled nanoparticle reporters (NPRs) in low binding tubes. The LoBs and the NPRs were each added to the reaction tube which was also a low binding tube. The reactions were incubated shaking for 20 min. Following incubation, the entire reaction volume (85 μL) was transferred to a polished aluminum surface where the LoBs were allowed to rise to the surface (approximately 5 min).4 We did not observe problems related to evaporation of the droplet in the ∼5 min time for LoB floatation and Raman collection. Data were acquired on a Snowy Range Instruments Sierra Raman instrument at 808 nm, rastering over a 2 mm × 0.5 mm area and acquired for 5 s. Five data points were acquired for each sample to establish a standard deviation. The Raman tag, BPE peak of 1600 cm−1 was normalized against the glass fluorescence peak at 1000 cm−1 to establish an internal standard. The intensity of the normalized peaks was plotted on a standard addition plot to arrive at the starting concentration and the apparent limit of detection for this assay. Data Acquisition and Analysis. Data were acquired on a Snowy Range Instruments Sierra Raman ORS instrument with an 808 nm rastering laser. By rastering the laser beam over a 2 mm × 0.5 mm area, the laser spot size remains small, which is a requirement for selectivity in Raman spectroscopy while a larger area is sampled allowing averaging of possible inhomogeneity. The SEM images in Figure 2 illustrate that with the current design, the LoBs appear to have locations where there are many and few NPRs. This problem is averaged out with the rastering laser. One of the signature peaks of each Raman tag was chosen for analysis (1600 cm−1 BPE), and 1000 cm−1 glass as an internal standard was chosen to standardize each data point. The internal standard was a fluorescence peak generated from the glass of the LoBs. The intensity of the peak from the Raman tag was divided by the intensity of the internal 4234
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enhancement shifts with the number of AuNPs and their orientation.14 Their assumption is that the large SERS signals observed from dimers and multimers stem from single molecules in the AuNP junctions. Our shelled NPRs also show a large number of dimers and multimers; Figure 2D has 3 monomers, 2 dimers, and 1 quadramer. Figure 4A is the spectrum obtained from 1 × 104 ng of CT in a LoB assay. The peak around 1000 cm−1 is due to
standard peak to arrive at a standardized intensity for each sample point. This eliminates variations in intensity due to differences in focus in individual samples. Data from each sample was acquired 5 times to ascertain the standard deviation of the LoB assay.
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RESULTS SERS Measurements. Our Raman measurements were made with an 808 nm Sierra Raman ORS system (Snowy Range Instruments). This system is capable of maintaining a high etendue with a tightly focused laser beam, yet it can be adjusted to examine a large sample area. We found that our LoBs were static and formed a monolayer at the top of the sample droplet, Figure 3. Our focused laser beam’s diameter
Figure 3. Schematic of the Raman measurement method used in our assay: (A) side view illustrating the spatial separation between LoBs and unconjugated AuNPRs and (B) top view illustrating further spatial separation between the focused LoBs and the dispersed AuNPRs.
was approximately 30 μm or about the size of one silica bubble. We performed a mock assay and obtained a micrograph of the bubbles. We counted the bubbles in the assay and found a monolayer of ∼1000 bubbles. In a monolayer, this equates to a diameter of 1 mm. We tuned our raster circuitry to produce a spherical pattern of slightly larger than 1 mm to capture the signal from all of the LoBs. The cholera assay was performed on a droplet placed on an aluminum surface to create a curved surface to focus the LoBs at the surface, see Figure 3A. The underlying concept is that the indirect LoB assay is to concentrate the positive assays, bubbles conjugated to shelled NPRs, and to separate the signals from the conjugated NPRs from the unconjugated. Our shelled NPRs have a density of 2.95 g/cm3, using 200 nm for the SiO2 shell diameter and 50 nm for the AuNP particle diameter. This causes them to rapidly sink and interfere with the results of a paramagnetic or centrifugal pull-down assay. Our optical method scans the top of the droplet and locates the positive LoBs. The focus of the beam and the opacity of the LoBs differentiates between the silica bubbles on top of the droplet and the material near the bottom. Figure 3B illustrates that the focusing of the particles will also produce a spatial differentiation as the unbound NPRs will disperse to a larger area in the sample. SEM analysis of the assay materials demonstrates that the assay consists of multiple AuNPs in each shell and that a single silica bubble binds with multiple NPRs, see Figure 2. An SEM/ Raman study by Wustholz et al. demonstrated that the local surface plasmon resonance (LSPR) responsible for the SERS
Figure 4. Assay results for CT: (A) Raman spectrum from 10 μg of CT pulled out with LoBs and NPRs. (B) Standard addition plot with calculated limit of detection.
luminescence from the silica bubbles. We observed this peak in silica with NIR excitation, and it is very strong with 808 nm excitation. We used this as an internal control to account for the number of LoBs at the droplet’s apex. This accounts for LoBs lost during the assay development and transfer to the sampling surface. The 1600 cm−1 peak stems from the reporter molecule, 1,2-bis(2-pyridyl) ethylene (BPE). Cholera detection is commonly required in water supplies or stool samples. Both cases present a complex sample matrix. Additionally, the CT antigen used for assay development contains stabilizers and preservatives that affect our assays. We used standard additions to account for interactions between the matrix and the analyte. Figure 4B is the standard addition graph obtained from our experiments. The value of the unknown is found by [c] = b/m
where [c] is the unknown concentration, b is the y-intercept, and m is the slope. The y axis in our plot is the ratio of the silica emission peak around 1000 cm−1 and our reporter molecule, BPE, peak around 1600 cm−1. Using this method and a linear regression, we found our predicted unknown to be 3700 ng (actual 5000 4235
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Technical Note
Notes
ng). The limit of detection (LOD) was found to be 1100 ng from the linear regressions predicted error in the y-intercept and the slope:
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank Snowy Range Instruments for the instrumentation and facility usage. Dr. Martoglio acknowledges the support of DePauw University for his sabbatical leave.
LOD = 3(σ /m)
where σ in this case is the predicted error in the y-intercept. This may slightly overestimate the LOD as the calculated predicted error in the y-intercept includes the errors of all the data and since we see significant heteroscedasticity in the data. However, the calculation provides a reasonable approximation. The heteroscedasticity is interesting. It is nearly 20 times larger than the predicted spectroscopic noise from the signals. We suggest that it is due to the variations in the signals due to loss of particles during the assay and the transfer of particles to the sampling surface. This error should be larger when the silica LoBs contain more NPRs. In other words, the loss of 10% of the highly positive LoBs will result in a larger error than 10% of a low positive assay. All results are discussed as mass rather than concentration since the buoyant LoBs enable us to detect mass independent of volume. The LoBs will concentrate on top of whatever volume is in the sample. We see this as a significant benefit as the concentration (analyte/volume) should be very low for samples with large volumes. Diagnostic assays are not commonly used in developing countries. Reagents are often refrigerated, trained personnel must operate the instruments, and much laboratory equipment is required to run diagnostic tests. The LoB platform for the sandwich assay frees the tests from any volume limitation that the magnet strength would dictate in traditional paramagnetic assays. It also decreases the likelihood of finding false positives from contamination of the sample to be interrogated with the NPRs. There are a number of reports of potentially commercial CT tests in the literature, but we found only assay, a lateral flow immunoassay (LFI), the SMART Cholera 01,15 which is actually commercially available. The Cholera 01 SMART II LFI reports an LOD at 2 × 107 colony forming units (CFU) per mL,15 and Spira and Fedorka-Cray found that there are approximately 0.19 fg/CFU Cholera toxin in Vibrio cholerae 01.16 This places their detection limit at 3.9 ng/mL of CT. While this appears to be much lower than our mass detection limit, we do have the advantage of detecting small levels of CT in large volumes. Additionally, this is proof of concept study and a report that has not been optimized for the number of LoBs, antibodies, or experimental conditions. Many research groups provide CT detection limits that fluctuate widely. This is not a comprehensive literature review, but a few CT detection limits are 1 nM CT on a biosensor,17 from 1 to 0.49 ng/mL using enzyme linked immunosorbent assays (ELISAs),18,19 sandwich (indirect) assays were reported at 40 ng/mL and 1 μg/mL while direct assays were reported at 200 ng/mL.20 Schofield et al. reported a detection limit of 3 μg/mL using glyconanoparticles in a colorimetric assay21 making their detection limit around 4 μg of Cholera toxin.
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REFERENCES
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AUTHOR INFORMATION
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[email protected]. Present Address §
Snowy Range Instruments, 628 Plaza Lane, Laramie, WY 82070. 4236
dx.doi.org/10.1021/ac300242k | Anal. Chem. 2012, 84, 4233−4236
