Anal. Chem. 2009, 81, 9824–9827
Detection of Protein Binding Using Activator Generated by Electron Transfer for Atom Transfer Radical Polymerization Hong Qian and Lin He* Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695 A purge-free controlled living polymerization method, activator generated by electron transfer for atom transfer radical polymerization (AGET ATRP), is used to amplify the occurrence of protein binding events. Detection of ovalbumin is demonstrated where binding of femtomole protein is differentiable from the background using ellipsometry. Moreover, binding of subpicomole protein leads to visually distinguishable spots on the sensor surface within 15 min, which signifies its potential in future development of point-of-need devices. Development of novel strategies to enhance detection of protein binding events is one of the most intensely investigated subjects in the sensing community for their potential in medical and environmental applications. It is particularly important for point-of-need portable sensing devices to be cost-effective and allow fast screening of minute amounts of analytes outside of sophisticated clinical or academic research settings.1-3 The commonly reported solutions for point-of-need sensing used in lateral flow (LF) devices or dipsticks rely on enzymatic reactions to improve assay sensitivity.4-6 Converting protein detection to DNA-based detection provides another solution that takes advantage of the polymerase chain reaction (PCR) for signal amplification.7,8 However, reliance on delicate enzymatic materials in both strategies imposes a major hurdle to convert research-lab concepts to commercially viable sensing products. Using specially designed probes to intensify protein binding events presents a possible answer to the challenge. Successful examples include the use of metallic Au nanoparticle networks to allow colorimetric readouts of protein binding events or the use of quantum nanocrystals with intense fluorescence emission, etc.9-11 Nevertheless, commercial adaptation of nanomaterials in the * Corresponding author. E-mail:
[email protected]. Phone: 919-515-2993. Fax: 919-515-8920. (1) Sapsford, K. E.; Bradburne, C.; Detehanty, J. B.; Medintz, I. L. Mater. Today 2008, 11, 38–49. (2) Wang, J. Biosens. Bioelectron. 2006, 21, 1887–1892. (3) Ligler, F. S.; Sapsford, K. E.; Golden, J. P.; Shriver-Lake, L. C.; Taitt, C. R.; Dyer, M. A.; Barone, S.; Myatt, C. J. Anal. Sci. 2007, 23, 5–10. (4) Pohanka, M.; Jun, D.; Kuca, K. Drug Chem. Toxicol. 2007, 30, 253–261. (5) von Lode, P. Clin. Biochem. 2005, 38, 591–606. (6) Shim, W. B.; Dzantiev, B. B.; Eremin, S. A.; Chung, D. H. J. Microbiol. Biotechnol. 2009, 19, 83–92. (7) Niemeyer, C. M.; Adler, M.; Wacker, R. Nat. Protocols 2007, 2, 1918– 1930. (8) Huang, Y.; Nie, X. M.; Gan, S. L.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. Anal. Biochem. 2008, 382, 16–22. (9) Yang, M. H.; Wang, C. C. Anal. Biochem. 2009, 385, 128–131. (10) Shen, D.; Meyerhoff, M. E. Anal. Chem. 2009, 81, 1564–1569.
9824
Analytical Chemistry, Vol. 81, No. 23, December 1, 2009
development of portable sensors progresses slowly with great caution, primarily because of the lack of understanding on the impact of large-sized nanoparticles on binding kinetics and thermodynamics, as well as practical concerns over the amount of detection tags to be incorporated onto the proteins without compromising the protein stability and bioactivity. The recently reported amplification-by-polymerization concept for DNA detection provides a promising alternative to circumvent the aforementioned hurdles in protein analysis.12-14 In this approach, a small chemical moiety is attached to the DNA detection probe prior to DNA hybridization, which introduces little interference to normal DNA activities. The onset of signal amplification is carried out after DNA hybridization is completed by triggering a synthetic radical polymerization reaction. Tens and hundreds of small molecules, i.e. monomers, as signal reporters are subsequently assembled into long chain polymers at the specific location where the chemical moiety is attached, i.e., where DNA hybridization occurred.12 Less than 3000 copies of short oligonucleotides have been successfully detected on a flat surface without using any enzymes.13 More importantly, growth of polymers on a highly reflective surface, e.g., Au, has resulted in a change of surface opacity, which can be visually distinguished from the background without sophisticated readout equipment, a prerequisite for the development of any portable sensing platform. Disengagement of signal amplification from biomolecular binding renders this amplification-by-polymerization strategy conceptually applicable to any biomolecular interaction. However, its applications in protein detection are still in its infancy,15 partially because of concerns over stability of protein complexes during polymerization. The requirement for an oxygen-free environment during radical polymerization, as reported in previous literature,12,13 further dampens the excitement over this approach. We have recently adapted activator generated by electron transfer for atom transfer radical polymerization (AGET ATRP) as an improved controlled/“living” radical polymerization reaction that eliminates the need for air-purging to remove oxygen.16 It uses a reducing (11) Chang, T. L.; Tsai, C. Y.; Sun, C. C.; Chen, C. C.; Kuo, L. S.; Chen, P. H. Biosens. Bioelectron. 2007, 22, 3139–3145. (12) Lou, X.; Lewis, M. S.; Gorman, C. B.; He, L. Anal. Chem. 2005, 77, 4698– 4705. (13) He, P.; Zheng, W.; Tucker, E. Z.; Gorman, C. B.; He, L. Anal. Chem. 2008, 80, 3633–3639. (14) Sikes, H. D.; Hansen, R. R.; Johnson, L. M.; Jenison, R.; Birks, J. W.; Rowlen, K. L.; Bowman, C. N. Nat. Mater. 2008, 7, 52–56. (15) Sikes, H. D.; Jenison, R.; Bowman, C. N. Lab Chip 2009, 9, 653–656. (16) Qian, H.; He, L. Anal. Chem. 2009, 81, 4536–4542. 10.1021/ac900959v CCC: $40.75 2009 American Chemical Society Published on Web 11/10/2009
Scheme 1. (A) Schematic Drawing of the AGET ATRP Reaction and (B) Major Steps for Polymerization-Amplified Protein Detection
agent, such as ascorbic acid, to remove oxygen in situ and regenerate the Cu(I) catalyst from its inert oxidative state (Scheme 1A).16 AGET ATRP has been successfully used in DNA detection where quantitative measurements of specific DNA sequences with better reproducibility, simpler assay procedure, and faster assay turnaround than those using conventional PCR-based DNA detection method has been demonstrated.16 In this report, we describe the application of this purge-free polymerization method in protein detection by launching dynamic macromolecular growth on a surface after protein binding occurs. Stability of protein complexes during polymerization is confirmed, and quantitative analysis of ovalbumin is examined. Qualitative detection of ovalbumin and insulin in a multiplexed array format is also demonstrated. EXPERIMENTAL SECTION Materials. Gold substrates (50-Å chromium followed by 1000-Å gold on a float glass) were purchased from Evaporated Metal Films (Ithaca, NY). 2-Hydroxyethyl methacrylate (HEMA, 99%) was purchased from Sigma-Aldrich and purified using an inhibitor remover-packed column to remove methyl hydroquinone inhibitor. l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), sulfo-Nhydroxysuccinimide acid (s-NHS), and biotin-N-hydroxysuccinimide ester (NHS-biotin) were purchased from Pierce (Rockford, IL). Concanavalin A (Con A), streptavidin, bovine serum albumin, ovalbumin, biotinylated insulin, R-casein, β-casein, bromoisobutyryl bromide, dimethylformamide (DMF), mercaptoundecanoic acid (MUA), diethyl ether, copper(II) bromide, ascorbic acid (AA), sodium acetate, NaCl, CaCl2, MnCl2, MgCl2, phosphate buffers, and 2-N-morpholinoethane sulfonic acid (MES) were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. Tris[(2-pyridyl)methyl]amine (TPMA) was purchased from ATRP Solutions (Pittsburgh, PA). Preparation of the Sensor Surface. Au substrates were cleaned in EtOH for 1 h prior to use. The cleaned substrates were immersed in a saturated mercaptoundecanoic acid (MUA)/EtOH solution overnight. After the MUA-coated Au surfaces were thoroughly rinsed with EtOH and H2O, the surfaces were activated with 50 mM EDC/15 mM s-NHS in 50 mM, pH 4.5 MES buffer for 2 h. Protein capture probes (ConA or strepta-
vidin at 1 mg/mL, 2 µL each) were drop-coated on a cleaned Au substrate in an array format, and the substrate was left in a humid chamber for 30 min. The surface was then rinsed with a phosphate buffer (pH ) 7), followed by incubating with 1 mg/mL BSA in 5% PEG for 30 min to block unreacted coupling sites and remove physically adsorbed protein probes. Meanwhile, the NHS-coupled ATRP initiator, N-hydroxysuccinmidyl bromoisobutyrate, was prepared following the previously reported procedure.12 The NHS-coupled initiator (10 mg/mL in DMF, 10 µL) was then added to the protein target solution (ovalbumin, BSA, or biotinylated insulin at 10 mg/mL) where the NHS-initiator-to-protein molar ratio was controlled at 10:1. The final resulting loading ratio of initiators on ovalbumin and BSA was confirmed by electrospray MS as 7-8:1. A ratio of 1:1 was achieved for insulin because of its smaller size. Next, NHS-biotin (10 mg/mL in DMF) was also added to the initiator-coupled BSA solution (10 mg/mL) to form biotin-initiator-labeled BSA conjugates. The total reaction volume of each solution was brought to 100 µL in the corresponding buffer, as specified in the text. All coupling reactions were finished within 1 h at room temperature. After centrifugal separation of initiator-labeled proteins from the unreacted ATRP initiators or NHS-biotin in the Eppendorf tubes with 3K molecular weight cutoff membrane, the purified protein solution, i.e., ATRP initiator-coupled ovalbumin and ATRP initiatorcoupled biotinylated insulin or BSA, was adjusted to 1 mg/mL and stored at 4 °C until needed. Before protein binding assays, a series of solutions were prepared by diluting ATRP-initiator-coupled ovalbumin in a 50 mM sodium acetate buffer with 0.1 M NaCl, 1 mM CaCl2, 1 mM MnCl2, 1 mM MgCl2, pH ) 5, to the desired final concentration. The stock solutionsofATRPinitiator-coupledinsulinandATRPbiotin-initiatorcoupled BSA were diluted in a 50 mM phosphate buffer, pH ) 7. The protein solution containing one or multiple target analytes at various concentrations was incubated with the spots where protein capture probes were immobilized for 30 min, followed by subsequent washes with 50 mM phosphate buffer. Surface-Initiated AGET ATRP Polymerization. In a typical surface-initiated AGET ATRP reaction, a mixture of CuCl2 (4.0 mg) and TPMA (8.7 mg) was added to 1.6 mL of HEMA solution (1:1 v/v HEMA/H2O). The initiator-immobilized substrates were then immersed into the reaction mixture, and the reaction vial (2 mL) was immediately sealed after 50 µL of ascorbic acid (AA, 96 mg/ mL) was added. Polymerization was run at room temperature for various periods of time, as specified in the text. The substrates were then rinsed in MeOH, followed by copious water wash. After the polymer was blown dry with nitrogen, the film thickness was determined by ellipsometry. Film thicknesses were measured with an AutoEL-III automatic ellipsometer (Rudolph Research, Hackettstown, NJ). The instrument irradiated the substrates at a 70° incident angle. All surface measurements were conducted on dried polymer films. RESULTS AND DISCUSSION Scheme 1B shows the major steps undertaken in AGET ATRPbased protein detection. Three protein complexes were selected as the models in the concept-proof experiment in which concanavalin A (Con A, probe A) and streptavidin (probe B) were used as the capture probes for specific detection of ovalbumin (target a), biotinylated insulin (target b1), or biotinylated BSA Analytical Chemistry, Vol. 81, No. 23, December 1, 2009
9825
Figure 2. A plot of polymer film thickness against the reaction time atop (0) Con A-ovalbumin or (O) streptavidin-biotinylated insulin. Both ovalbumin and insulin were preconjugated with ATRP initiators. The inset shows the zoom-in of time-dependent polymer growth atop insulin. Target protein concentration ) 1 mg/mL. The error bars were calculated based on three replicates.
Figure 1. (A) A photoimage of 1 mg/mL target protein detected after 15-min AGET ATRP. Polymer growth results in the directly observable opaque spots (Con A/ovalbumin), whereas the control spot remains transparent (streptavidin/ovalbumin). The dotted circle is merely a guide for the eyes. (B) Ellipsometric measurements of the film thicknesses after polymer growth where different protein molecules were mixed. The x-axis indicates the target protein molecules in the incubation solution, and the columns of different colors/patterns refer to the protein capture probes immobilized on the surface. The error bars were calculated based on three replicates.
(target b2), respectively. Both protein capture probes (1 mg/mL) were immobilized to a mercaptoundecanoic acid-coated Au surface in the presence of EDC/NHS. Subsequent surface blocking using unmodified BSA in a 5% PEG solution was conducted to react with residual active carboxyl groups, passivate the Au surface, and remove physically adsorbed protein capture probes. Meanwhile, excess ATRP initiators (NHS ester of bromoisobutyroyl bromide) were reacted with the primary amino groups on ovalbumin, biotinylated insulin, and biotinylated BSA.12 The final loading ratio of the initiators per ovalbumin was quantitatively determined by electrospray mass spectrometry (data not shown) and was found to be consistent with the feeding ratio of the coupling reagents. After centrifugal separation of initiator-labeled proteins from unreacted ATRP initiators using a centrifuge tube with 3K-Da molecular weight cutoff membrane, the purified protein solutions, i.e., ATRP initiator-coupled ovalbumin and ATRP initiator-coupled, biotinylated insulin and BSA, were adjusted to 1 mg/mL before incubating with the capture protein-coated surfaces for 30 min. The substrate was thoroughly rinsed with 50 mM phosphate buffer before being loaded into an AGET ATRP reaction mixture of CuCl2, tris[(2-pyridyl)methyl]amine (TPMA), and hydroxyethyl methacrylate (HEMA, 1:1 v/v in H2O). The reaction vial was immediately sealed after 50 µL of ascorbic acid (AA, 96 mg/mL) was added. Polymerization was run at room temperature and stopped when the substrate was taken out and rinsed in methanol to remove excess HEMA. Direct Au surface inspection showed the change of substrate reflectance at the spots where expected protein binding occurred, suggesting the survival of protein complexes in AGET ATRP and the successful formation of organic films upon protein binding (Figure 1A). Ellipsometric measure9826
Analytical Chemistry, Vol. 81, No. 23, December 1, 2009
ments show that within a 15-min reaction, more than 45 Å in positive film growth was obtained atop the Con A/ovalbumin spot, ∼9 Å atop the streptavidin-biotinylated insulin pair, and 38 Å atop the streptavidin-biotinylated BSA pair (Figure 1B). In comparison, the controls, i.e., the spots of streptavidin/ovalbumin, Con A/biontinylated insulin, and Con A/biontinylated BSA, had much less materials accumulated. More effective polymer film growth at the spots of Con A/ovalbumin and streptavidin/biotinylated BSA is attributed to the larger molecule size of both proteins over insulin (45 kDa and 67 kDa, respectively, vs 5.8 kDa of insulin). The larger target size allowed a higher initiator coupling ratio, which subsequently led to more polymer chains grafted per binding event, i.e., thicker polymer films measured. AGET ATRP is a controlled/living polymerization reaction where the polymer film thickness theoretically increases with extended reaction time until all monomers are consumed. However, the high initiator density on the protein molecules could result in increased chain transfer or chain termination that slows polymer growth. As shown in Figure 2, polymer chain growth slowed and reached a plateau in 2 h. In contrast, for smaller proteins with fewer initiators, such as insulin in this case, the polymer growth was less affected and the living characteristics were preserved. This quasiliving grafting of polymer chains is the basis for the tunable feature of the amplification-by-polymerization platform where the degree of signal amplification is adjustable by changing the reaction time based on the estimated analyte concentration. For example, Figure 2 shows that a 30-min reaction was sufficient to grow polymer films well above the detection background when the analyte concentration was in excess (1 mg/mL). When the concentration of the protein target decreases, however, the fewer polymer chains grafted on the surface due to fewer binding events can be easily compensated by extending the reaction time and growing each polymer chain longer. Figure 3 shows quantitative detection of ovalbumin when its concentration varied. The thickness initially changed linearly, but the growth slowed when the binding of target molecules to the surface capture probes approached to saturation. A regression fitting in the low concentration range (0-100 µg/mL) yields a linear fitting with a correlation coefficient R2 of 0.95. Approximately 46 Å in positive film growth was measured from the spot incubated with 0.1 µg/mL ovalbumin, well above the background signal of 17 ± 5 Å. This detection limit equals ∼5 fmol (i.e., low ng/mL)
Figure 4. A photographic picture of polymer growth on an arrayed substrate after AGET ATRP polymerization. Surface capture proteins (left) and target analytes (bottom) are labeled. Target protein concentration in the protein mixture ) 1 mg/mL. ATRP reaction time ) 30 min.
the presence of the specific analyte; the position of the spot illustrates the nature of the analyte detected. Figure 4 shows the concept-proof experiment for an arrayed assay: two target analytes, ovalbumin and biotinylated BSA, separately or together, were spiked in a protein cocktail solution containing R-casein, β-casein, and unmodified BSA. The color change at spot a indicates the presence of ovalbumin in solution 1 whereas the color change at spot d suggests positive detection of biotinylated BSA in solution 2. Both spots lighten up when both analytes were present in the solution 3. Among three replicates conducted, zero cross-identification, i.e., false positive or false negative, was observed.
Figure 3. (A) A photographic picture of an array of Con A-modified spots incubated with initiator-coupled ovalbumin at different concentrations. The arrows labeled the target protein concentrations as well as the expected incubation position. (B) A logarithmic plot of polymer film thickness as a function of ovalbumin concentrations. ATRP reaction time ) 1 h. The error bars were calculated based on three replicates.
ovalbumin detected, comparable to what was reported for traditional immunochemical methods but yet to reach the level achievable by enzymatic amplification. Further assay optimization to reduce background from nonspecific adsorption and extension of polymerization time to allow grafting of longer polymer chains could lead to more competitive detection sensitivity. It is important to note that close inspection of the array of spots incubated with different concentrations of ovalbumin shows the spot clearly distinguishable from the background when the concentration of target protein was as low as ∼10 µg/mL (Figure 3A), equivalent to subnanogram or picomole amount of materials visually detected. The spot became less discernible when the protein concentration was further reduced to 1 µg/mL, despite positive film growth still measurable with ellipsometry. Localized AGET ATRP makes the reaction amenable to protein arrays where different capture probes are differentiated from each other by their physical positions. Development of a spot with a lighter color against the background suggests positive polymer growth, i.e.,
CONCLUSION In summary, the amplification-by-polymerization concept has been successfully demonstrated in protein binding detection. Adaption of the AGET ATRP reaction eliminates the purging step and allows fast polymer growth for which subnanogram amounts of proteins are directly visualized in less than 15 min. The limit-of-detection of the method at the current stage is comparable to traditional immunochemical assays but is yet to be competitive to ELISA assays. Extending reaction time or using bulkier monomers can potentially improve detection sensitivity. Optimization of reaction catalysts, demonstrated in the past, has a higher impact on the polymerization reaction kinetics and can also improve detection sensitivity if needed. Applying the amplification-by-polymerization sensing concept to sandwich immunoassays is currently underway. ACKNOWLEDGMENT Assistance from Mr. Yongsheng Xiao on electrospray mass spectrometric characterization of the initiator-labeled proteins is acknowledged. This work is partially supported by NSF (No.0644865).
Received for review May 4, 2009. Accepted October 29, 2009. AC900959V
Analytical Chemistry, Vol. 81, No. 23, December 1, 2009
9827
