A Streptavidin-SOG Chimera for All-Optical ... - ACS Publications

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A Streptavidin-SOG Chimera for All-Optical Immunoassays Elizabeth M. Wurtzler and David Wendell* Department of Biological, Chemical, and Environmental Engineering, University of Cincinnati College of Engineering and Applied Science, 2901 Woodside Drive, Cincinnati, Ohio 45221, United States S Supporting Information *

ABSTRACT: Immunological detection has been developed into a sensitive and versatile technique and is based on two requisite elements: targeting antibodies and an indicator, usually in the form of horseradish peroxidase or alkaline phosphatase. The specificity and turnover rate of these enzymes provide an efficient means of signal amplification, but both require a stopping agent to prevent overdevelopment, which limits scalability to mechanized fluidic systems. As an alternative, we present a fully optical detection system based on a streptavidin-singlet oxygen generating chimeric protein that produces singlet oxygen from blue light. When used with trans-1-(2′-methoxyvinyl)pyrene, the photosynthetic streptavidin combines indicator development and reporting in sufficiently distinct visible wavelengths while retaining the sensitivity and scale of enzymatic systems that use horseradish peroxidase. By combining photosensitive development and detection into one system, we can enable future, highly parallel immunological testing to be controlled with the spatial and temporal precision of light.

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INTRODUCTION In the last four decades, immunological detection has been developed into a sensitive technique for a variety of applications ranging from disease screening1,2 to toxin testing.3−5 Immunological methods require two detection elements: antibodies and an indicator agent, usually in the form of horseradish peroxidase or alkaline phosphatase. The specificity and turnover rate of these enzymes provide an efficient means of signal amplification; however they also require an additional liquid handling step in order to prevent overdevelopment. The mechanization required for liquid handling and potential for nonspecific development limit the scalability of peroxidase-based immunoassays. Here we present a fully optical analyte detection method that combines indicator development and reporting in sufficiently distinct visible wavelengths while retaining the simplicity, reactivity, and scale of enzymatic systems such as HRP. When combined with antibodies, this approach lends itself to a substantially parallel and automated system by combining both development and detection phases of the assay via light allocation. A photometric immunoassay with cost-effective throughput would enable increased access to a host of critical biological and medical applications, such as HIV and cancer screening. Traditional enzyme-linked immunosorbent assays (ELISA), have relied on antibodies conjugated either directly or indirectly to HRP as the primary means of colorimetric detection. The choice of HRP has been linked to the size, stability, and cost advantages of this enzyme over alternatives like alkaline phosphatase. Similarly, HRP-streptavidin conjugates are commonly used for generic detection of biotinylated antibodies because of the larger variety commercially available. The peroxidase-streptavidin pairing with biotinylated antibodies exploits the specificity and near covalent strength of the biotin© 2013 American Chemical Society

streptavidin interaction to deliver the developing agent in a rapid, targeted manner.6,7 A variety of HRP signal substrates are commercially available, such as diaminobenzidine (DAB), 3,5,3′,5′-tetramethylbenzidine (TMB), and 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS). These substrates provide an absorbance maximum upon HRP oxidation allowing for convenient spectrophotometric detection of antigens. Despite the efficient reaction rate of HRP, detection is limited by the requisite reaction termination agent for this enzyme which prevents undesirable assay overdevelopment. In addition to the required liquid transfer and handling process, the given choice of substrates do not always provide adequate sensitivity. Chemiluminescent substrates for HRP have been shown to increase the sensitivity of detection;8 however, these substrates remain below adequate quantum yields (1.5% in aqueous solution),9,10 requiring enhancers for sufficiently luminous signal lifetimes.11,12 In order to avoid the limitations of peroxidase enzymes and the corresponding substrates, lanthanide-antibody conjugates using Europium have been developed.13,14 These conjugates have large Stokes shifts (up to 290 nm)15 and fluorescent lifetimes as long as 1 ms providing adequate signal-to-noise and a convenient means of optical reporting. Similarly, antibodies or streptavidin have been simultaneously conjugated to nanoparticles and lanthanides for use in time-resolved immunofluorescence assays with the intent of improving sensitivity by affecting an increase in labeling efficiency.16,17 Lanthanides have additionally been conjugated to streptavidin for direct detection of biotinylated antibodies.6 However, these conjugates require complex chemical labeling Received: October 10, 2013 Revised: November 26, 2013 Published: December 3, 2013 228

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Chemicals). E.coli BL21(DE3) cells and Overnight Express Medium (EMD Chemicals) were used for protein expression. Cells were resuspended in a 50 mM Tris-HCl, 300 mM NaCl buffer, pH 7.8, and lysed via French Press. Cell lysate was cleared by centrifugation at 30 000g, 4 °C for 30 min. Protein was purified on His60 Superflow Resin (Clontech) by washing with a 50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole buffer, pH 7.8, and eluted with a 50 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole buffer, and pH 7.8. Generally, a yield of 5−10 mg of protein was obtained per liter. Purification was verified via Native and SDS PAGE gels (Supporting Information Figures S1 and S2, respectively). Prior to use, imidazole was selectively removed from the buffer via dialysis using 20KMW Slide-a-lyzer cartridges (Pierce) and 3, 1 L exchanges against washing buffer. Genes for nonstreptavidin conjugates consisting of only tandem and triplet SOG domains (TanSOG and TriSOG respectively) were de novo synthesized by Operon. A 6× His tag was adjoined to the C-terminus of both constructs and a StrepII tag was added to the N-terminus of the triplet SOG. Both constructs were inserted into pET51b by ligation independent cloning. Protein expression and purification was performed as previously described. Binding constants for SMS were determined by fluorescent quenching as described previously35 with the exception that optical excitation of 450 nm wavelength was used for koff measurements to provide a FRET transfer at 475 nm wavelength to fluorescein, monitoring emission at 525 nm. For kon, 100 pM biotin-4-fluorescein in 180 μL of PBS (pH 7.4) was added to a 20 μL volume of 17.87 nM SMS in PBS. Quenching of the biotin-4-fluorescein due to binding to SMS was determined with optical excitation at 496 nm wavelength and emission at 521 nm wavelength. For koff, a solution of 12 nM biotin-4fluorescein and 0.12 mg/mL BSA in 170 μL of PBS was added to 1 μM SMS and was incubated in the dark at room temperature for an hour. After incubation, 1 mM unlabeled biotin in PBS was added to the solution. Fluorescence of the challenged biotin-4-fluorescein being released from SMS was monitored at 525 nm. Excel was used for linear regression to determine koff from the plot of ln(fraction bound unlabeled biotin) against time. All readings were taken in the FlexStation3 Plate Reader (Molecular Devices, CA) every 5−7 s for 1 h at room temperature. In order to measure singlet oxygen generation rates, SMS, TanSOG, and TriSOG (29.3 μM) were separately added to a 50 mM Tris-DAB (0.25 mg/mL) solution (pH = 7.6) and irradiated with LED generated 450 nm light (28 μmol m−2 s−1, Luxtec ACO Series 8000, Luxtec Corporation) for 1 h while measuring absorbance at 595 nm (n = 4). For dot blots with DAB, 5 μL of spots of biotinylated β-galactosidase (β-gal) in amounts of 2.5 μg, 250 ng, 25 ng, and 2.5 ng were deposited and allowed to fix on 0.45 μm of nitrocellulose and covered with optically opaque shielding. β-gal was chosen for its ability to provide an alternative means of detection through X-gal development, a feature not utilized due to limited experimental use and sufficient DAB polymerization with SOG. Equal volumes and amounts of BSA and SMS were used as positive and negative controls, respectively. Unbound portions of the membrane were blocked by incubating with 5% skim milk (Difco) for 1 h at room temperature and then washed through three cycles with PBS pH 7.4. A quantity of 0.5 mg of SMS was added to the membrane and incubated at room temperature for 1 h and the membrane was again washed for three cycles with PBS. The membrane was then immersed in a DAB solution of 0.25 mg/mL and exposed to 450 nm light (28 μmol m−2 s−1, Luxtec ACO Series 8000, Luxtec Corporation) for 15 min. For the dot blots with trans-1-(2′-methoxyvinyl)pyrene (MVP, Life Technologies), 5 μL of spots of biotinylated β-gal in amounts of 2.5−15 ng were deposited and allowed to dry on nitrocellulose membranes corresponding to the positions of the wells on a 96-well plate and covered with optically opaque shielding. The membrane was blocked by incubating with 5% skim milk at room temperature for 35 min. The membranes were washed through three cycles with PBS. One hundred nanograms of SMS was added to the wells and incubated at room temperature for 35 min and the membranes were again washed for three cycles with PBS to remove unbound SMS. A quantity of 10 μM MVP in H2O was then added to the membrane and irradiated with 450 nm light (460 μmol m−2 s−1, “All Blue LED panel”, ERZ Industries) for 35 min,

processes, are more expensive, and are limited by reduced diffusivity relative to other complexes. Indeed, comparing our streptavidin complex to these lanthanide conjugates using the Stokes−Einstein equation (see Supporting Information Equation S1) and radii estimates from molecular weight,18 the relative one-dimensional diffusivity is less than half. Further, toxicity of lanthanide compounds remains a concern for cellular immunoassays as lanthanide chelates have been found to induce apoptosis via DNA fragmentation19 and bind to Ca2+ sites on cell membranes,20 inhibiting critical ATPases.21 For more than three decades, streptavidin has been used for biotinylated protein detection, either directly or indirectly via antibody mediated targeting strategies. Improvements to streptavidin-mediated protein development have focused on conjugates including such reactive approaches as emission from quantum dots,22−24 luciferase,25−27 and lanthanides.6,7 Regarding the former two strategies, it remains unclear whether the resulting large conjugates affect biotin binding, particularly through steric hindrance and/or pore occlusion as it has yet to be thoroughly evaluated. Additionally, expression and purification of streptavidin fusion proteins has continued to pose difficulties because the protein must be extracted from inclusion bodies,27 coexpressed with a chaperone plasmid,26 or expressed in eukaryotic cells.25 In order to overcome the aforementioned limitations and enable the most efficient hybrid of fluorescence activity and developmental attributes, we have selected the recently created Light-Oxygen-Voltage 2 domain mutant from Arabidopsis thaliana referred to as singlet oxygen generator (SOG).28 SOG is a prime candidate as a developmental indicator due to its superior optical properties including green fluorescence and singlet oxygen production at a quantum efficiency of 0.47.28 Our construct consists of the SOG domain attached to the c-terminal end of streptavidin via a flexible amino acid linker. The triplet glycine linker provides sufficient plasticity for proper SOG folding but also enough restriction to ensure SOG does not occlude the streptavidin−biotin-binding pocket, a property first modeled in PEP-FOLD29,30 and then verified using biotin-4fluorescein dissociation testing. The SOG domain was also chosen to assist streptavidin solubility in E. coli and was allowed for robust yields without the need for denatured inclusion body purification and refolding as traditionally required.31−33 An additional advantage for our developmental indicator is the relatively short lifetime of singlet oxygen in aqueous solutions (2 μs),34 which limits the diffusional distance and nonspecific interactions, promoting high signal-to-noise ratio and preventing undesirable nontarget development. This streptavidin-SOG conjugate, which we call Strep-Mini-SOG (SMS), can be controlled with the temporal and spatial precision of tunable, optical exposure and universally applied to biotinylated antibodies without the need for consumable energetic substrates (i.e., luciferin) or reaction termination processes.

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EXPERIMENTAL SECTION

Materials and Methods. All reagents were purchased from SigmaAldrich unless otherwise noted. The streptavidin in pET21a (Plasmid 20860) was obtained from Addgene. The SOG construct was de novo synthesized by Genscript. PCR was used to synthesize the requisite streptavidin and SOG gene with the addition of a 3 amino acid linker, 3× Gly, and a 5× His tag added to the C-terminus of the chimeric construct for purification. The Gly linker simulations were performed on PEPFOLD29,30 using 3−8 flanking amino acids from the streptavidin ctermini and SOG n-termini in equal proportions. The SMS gene was inserted into pET51b by ligation independent cloning (EMD 229

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Figure 1. An illustration of the Streptavidin tetramer with c-terminal SOG domains attached. Both streptavidin (1STP)39 and SOG (A. thaliana LOV2, 4EEP40) were rendered in RCSB protein workshop. The streptavidin structure shows biotin within the binding pocket.

Figure 2. (A) Amount of biotin-4-fluorescein bound to SMS after addition of unlabeled biotin. Points represent an average of measurements taken at four consecutive time points (n = 4). (B) Amount of biotin-4-fluorescein dissociated from SMS after addition of unlabeled competitor biotin (n = 3). Error bars represent standard deviation. after which fluorescence was measured in the Flexstation using an excitation of 366 nm wavelength and emissions of 420 and 460 nm wavelengths.

produce a temporal signal amplification through singlet oxygen mediated degradation of MVP. Previous work describing the generation of H2O2 by antibodies in the presence of singlet oxygen36,37 indicates that conjugation of an SOG domain directly to an antibody could also improve signal generation and be useful in applications where a biotinylated primary antibody is undesirable. SMS Linker Design. In order to ensure unobstructed biotin binding after the addition of the SOG domain, we simulated the different conformational states of the triplet Gly linker along with the adjoining ends of streptavidin and SOG in PEP-FOLD.29,30

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RESULTS AND DISCUSSION Immunoassays are reliant upon reactive oxygen species for development, most commonly produced through enzymatic conjugates. Having already proven useful for protein specific labeling on the molecular scale,28 our system has extended SOG in the form of SMS to macroscale use as an optical visual indicator, both with internal green fluorescence and its ability to 230

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Protein Detection with DAB. Biotinylated β-gal was detected using SMS mediated DAB polymerization. Using direct observation, 2.5 μg of biotinylated β-gal was readily detected after 1 min exposure and 250 ng of biotinylated β-gal was detected after 5 min total exposure time (Supporting Information Figure S4). However DAB-based detection was limited by nonspecific oxidation, since DAB mixtures in Tris buffer were observed to autopolymerize over time, even when frozen. As a result, we tested MVP as optical indicator alternative. Protein Detection with MVP. Biotinylated β-gal was detected using the fluorescence shift observed upon 1O2 interaction with MVP. The increase in fluorescence at 460 nm resulting from the generation of 1-pyrenecarboxaldehyde (PCA) as a product of the reaction of singlet oxygen and MVP relative to the fluorescence at 420 nm from MVP was used for the detection of protein. Using this method, biotinylated β-gal was successfully detected in amounts ranging from 2.5 to 15 ng (Figure 4) using

Glycine was purposefully chosen to allow flexibility, which can be seen in the five models produced in the simulation (Supporting Information Figure S3). Overall, the protein termini can be seen to overlap, indicating that the ends would remain in close proximity and on the opposite side from the biotin binding pocket as shown in Figure 1. The Figure 1 illustration shows the relative domain sizes derived from the crystal structures for streptavidin (1STP)38 and SOG (4EEP).39 Biotin Binding Kinetics. FRET response was utilized in order to minimize background signal from the fluorescence of SMS, which has a similar emission output to biotin-4-fluorescein. The off rate, koff, at 25 °C and pH 7.4 was 2 × 10−5 s−1, similar to previously reported values for streptavidin using the biotin-4fluorescein method35 (Figure 2A). After an hour, only 9% dissociation was observed (Figure 2B). The on rate, kon, at 25 °C and pH 7.4 was 7.38 × 106 M−1 s−1 (n = 4), identical to values previously reported values using the biotin-4-fluorescein method35,40,41 Singlet Oxygen Generation Rates. Singlet oxygen generation rates of SMS, TanSOG, and TriSOG were determined via direct measurement of DAB polymerization rates42 (Figure 3). Surprisingly, singlet oxygen generation rates

Figure 4. Detection of biotinylated β-gal using the fluorescence of the product of MVP catalysis. MVP catalysis is defined as the fluorescent ratio of PCA/MVP with peak emissions at 460 and 420 nm, respectively. Error bars represent standard deviation (n = 5). Figure 3. Rates of singlet oxygen generation were found to be 2.9 × 10−3 min−1 (r2 = 0.996), 1.6 × 10−3 min−1 (r2 = 0.998), and 6.0 × 10−4 min−1 (r2 = 0.986) for SMS, triSOG, and tanSOG, respectively, as measured by increased absorbance at 595 nm due to DAB polymerization. Error bars represent the standard deviation (n = 4).

an exposure time of 35 min, a value optimized for the lowest protein concentrations. MVP development with larger amounts of protein was on par with the developmental speed of the DAB dot blot (5 min) and proportional signal increases with time (Supporting Information Figure S5). Using SMS and MVP as a means of analyte detection, protein was distinguished in amounts of 2.5−15 ng. These values are within the range of other streptavidin conjugate reporters27 and are consistent with detection limits of HRP linked antibodies.44 One advantage of pairing MVP with SMS is the equivalence of emission wavelength of catalysis product of MVP and the excitation wavelength of SMS generating a positive feedback amplification of the resulting signal. Spontaneous DAB polymerization was observed in Trisbuffered solutions maintained at room temperature and frozen at time scales similar to those used for immunoassay development protocols. As a result, DAB was found to generate undesirably high levels of background polymerization and was therefore eliminated as an appropriate indicator for spectrophotometric investigations. In contrast, MVP generated a high signal-to-noise ratio as well as minimal spectral overlap with SMS, enabling

were found to increase nonlinearly relative to attached SOG count. The DAB polymerization rates were found to be 2.9 × 10−3 min−1, 1.6 × 10−3 min−1, and 6.0 × 10−4 min−1 for SMS, triSOG, and tanSOG respectively. More specifically, these rates represent the DAB polymerization per SOG domain since sample dilutions were designed to maintain an identical total number. Thus, SMS has a rate 4.8 times higher than tanSOG, despite the same total number of SOGs in each reaction, indicating enhanced quantum efficiency for SMS. The increase in quantum efficiency from closely spaced fluorophores is indicative of a Förster resonance energy transfer mechanism since the SOG conjugated domains are within 10 nm of each other.43 The observed enhancement of singlet oxygen production was subsequently used to improve the sensitivity of immunoassays and decrease required development time. 231

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detection of at least 2.5 ng of analyte within the current limits of HRP immunoassays.44−46

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CONCLUSIONS An SMS-based analyte detection method would provide the opportunity for a large scale, cost-effective, solely optical detection system. Because the signal is light dependent, overdevelopment can be avoided by creating a feedback loop between excitation and detection, allowing for intrinsic automated development. In so doing, precise temporal control is permitted, allowing the ability to cyclically start and stop the reaction at will until the desired development is achieved. A future version for this system could eliminate the need for a partitioning structure, as is presently used in the 96-well plate, leaving only the immunoassay membrane to be developed differentially by a spatially tuned light array such as a DLP chip. Using a fluorescent signal to detect the presence of target proteins allows for precise and parallel indicator development while minimizing inefficient liquid handling protocols, thus reducing time and mechanistic requirements such as fluidic transfer systems. Taken together, SMS presents an opportunity to improve the throughput and cost efficiency of conventional immunoassay detection systems. Future work will explore improved quantum efficiency using concatenated SOG domains on the SMS tetramer. The binding, fluorescent and photosynthetic properties of SMS make it a valuable candidate for a variety of other applications including photolithography, microscopy and light-based disinfection.

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ASSOCIATED CONTENT

S Supporting Information *

Equations, PEP-FOLD structure simulations, protein gel electrophoresis images, and DAB dot blot results are included. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (513) 556-2482. Fax: (513) 556-4162. Author Contributions

The manuscript was written by E.W. and D.W. and experiments were performed and analyzed by both authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank J. Todd and L. Marquardt for reviewing the manuscript and for helpful discussions. This work was supported by funds from the College of Engineering and Applied Science at the University of Cincinnati. The authors declare no competing financial interest.

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REFERENCES

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