Progress 09/01/22 to 08/31/23
Outputs
Target Audience:The target audiences include 1) chemists and food scientists in the research areas of bioanalytical chemistry and biosensors, food safety, and polymer science, 2) food manufacturers in industry, 3) consumers with food allergy. Changes/Problems:The graduate student left from group due to graduation, which caused some delay for research progress. The first no-cost extension have been approved to 08/31/2024 and the second NCE could be possibly requested if needed. What opportunities for training and professional development has the project provided?One graduate student and one undergraduate student have been trained to use AbP in biosensing for protein detection, including DESIGNS OF BSA-BIOTIN-STREPTAVIDIN MODEL SYSTEMS, IMMOBILIZATION AND ACTIVATION OF SMALL MOLECULES, STREPTAVIDIN (SAv) AND CHAIN TRANSFER AGENT (CTA) COUPLING, IMMOBILIZATION OF SAv AND BSA/BIOTIN/SAv SANDWICH SYSTEM, and AMPLIFICATION-BY-POLYMERIZATION FOR BIOTIN DETECTION. The students have also been trained to use different instrumentation such as an alpha-SE ellipsometer (J.A. Woollam) for measurement of polymer growth thickness on the gold surface, Asylum MFP-3D Origin+ AFM for measurement of surface topography. How have the results been disseminated to communities of interest?USDA NIFA Food Safety & Defense Annual Grantees Conference, Toronto, Canada, 2023. He, P. "Polymerization-Mediated Amplification in a Sandwich Immunoassay for Protein Detection" Poster Presentation, International Association for Food Protection (IAFP) 2023 Annual Meeting, Toronto, Canada. Williams-McCord, K.; He, P. "Amplification-by-Polymerization in Protein Biosensing" Poster Presentation, Southeast Regional Meeting of the American Chemical Society (SERMACS) 2023, Durham, North Carolina. What do you plan to do during the next reporting period to accomplish the goals?proof-of-concept of peanut allergenic protein detection using reversible-deactivation radical polymerizations
Impacts
What was accomplished under these goals?
We continued to work on BSA-biotin-streptavidin model to optimize the experimental conditions for Amplification-by-Polymerization (AbP) in protein biosensing. For four different systems we investigated, we compared their performances and got good spot visibilities and reproducibilities for all. Then, we focused on System 3 which is closest to the protocol used for coming peanut allergen detection. 1) Comparison between with and without 6-mercapto-1-hexanol (MCH) surface passivation Without MCH, the reaction spot is 35.59 nm, the control is 4.56 nm, S/N is 7.80. With MCH, the reaction spot is 79.91 nm, the control is 6.17 nm, S/N is 12.95. Therefore, MCH surface passivation improved S/N. 2) Sensitivity test We have detected target biotin at 1.5 pg level, which equals to 1 ng/mL, with S (visible, avg 24.00 nm)/N (invisible, avg 3.99 nm) 6.02. We summarized here: a dynamic range from 1.5 pg (1 ng/mL) to 1.5 µg (1.0 mg/mL) with visible results compared with 1.5 fg (1 pg/mL) and control. This result can be comparable to ELISA technique. We used AFM (atomic force microscopy) to characterize the surface morphology of sensing surfaces before and after AbP.
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:The target audiences include 1) chemists and food scientists in the research areas of bioanalytical chemistry and biosensors, food safety, and polymer science, 2) food manufacturers in industry, 3) consumers with food allergy. Changes/Problems:Due to research delay caused by pandemic, the team will ask for no-cost extension for one more year to complete Objective 2 sensitivity, specificity, and accuracy optimizations for the developed biosensing method validation, performance comparison with commercially available ELISA kits, and further development into a multiplexed bioassay. What opportunities for training and professional development has the project provided?One graduate student has been trained to use AbP in biosensing for protein detection, including DESIGNS OF BSA-BIOTIN-STREPTAVIDIN MODEL SYSTEMS, IMMOBILIZATION AND ACTIVATION OF SMALL MOLECULES, STREPTAVIDIN (SAv) AND CHAIN TRANSFER AGENT (CTA) COUPLING, IMMOBILIZATION OF SAv AND BSA/BIOTIN/SAv SANDWICH SYSTEM, and AMPLIFICATION-BY-POLYMERIZATION FOR BIOTIN DETECTION. The student has also been trained to use different instrumentation such as an alpha-SE ellipsometer (J.A. Woollam) for measurement of polymer growth thickness on the gold surface, Asylum MFP-3D Origin+ AFM for measurement of surface topography. How have the results been disseminated to communities of interest?USDA NIFA Food Safety & Defense Annual Grantees Conference, Virtual Meeting, 2021. What do you plan to do during the next reporting period to accomplish the goals?proof-of-concept of peanut allergenic protein detection using reversible-deactivation radical polymerizations
Impacts
What was accomplished under these goals?
The year 1 implementation of the current USDA project has successfully demonstrated and designed four different model systems based on the amplification-by-polymerization (Abp) of the BSA-Biotin-Streptavidin binding system, which served as a proof-of-concept for the actual visual detection of peanut allergens. This initial investigation is vital and important to show the possibility and future success of the aforementioned project. Detailed information on each major activity and significant results are shown below. Streptavidin is one of the most well-studied proteins and has been used in many studies such as its application as a macroinitiator for the formation of polymer-protein conjugates. 14 Streptavidin is a protein with four subunits in which each of which is capable of binding with one molecule of biotin with a very high affinity of Kd = 10-15 M. 15 The modification of streptavidin with small molecules is vital to achieving a polymer formation that can be used as sensing platform via amplification-by-polymerization for the detection of biotin. Here, streptavidin was modified with the CTA to regulate and control the formation of the polymers. CTA is an important component in RAFT polymerization. It confers a living character to polymerization as it mediates the process via a reversible chain-transfer process. One mole of streptavidin was coupled with 10 moles of small molecule CTA-NHS. Considering the large size of streptavidin, the use of a 10th-fold amount of CTA is important to secure good polymer growth. The CTA's reactive NHS group covalently reacted with the amine groups of the streptavidin forming a SAv-CTA conjugate. Scheme 2A illustrates the first model system via Au/SAv-CTA binding. The CTA-coupled streptavidin here sits on top of the gold substrate via physical adsorption and limited covalent binding. The presence of polymer spots on the gold substrate after polymerization confirms a successful process that can be used as a sensing platform for visual detection. The Au/SAv was used as one of the negative controls which gave no spots after the polymerization because of the absence of the CTA in the streptavidin. CTA is an important component in RAFT polymerization in which without its presence, the surface-initiated polymerization will not occur thus no polymer spots will form on the gold surface. Scheme 2B shows the second model system via the Au/BSA/Biotin/SAv-CTA sandwich system, which reflects the actual detection of the target of interest, which in this case is the biotin. Since this is a proof-of-concept of the peanut allergen detection, the BSA herein assumes to be the primary antibody, biotin to be the peanut allergen, and streptavidin to be the secondary antibody. BSA has been used in many studies as a surface-blocking agent and thus can be used as the protein to cover the surface. The biotin as a target of interest can freely react with the BSA via non-covalent interactions. As streptavidin has a very high affinity with biotin, these bindings can be used to show the feasibility of these model systems for detection. Like the first system, the presence of polymer spots on the gold substrate after polymerization confirms a successful detection of the biotin target while the absence of spots indicates no detection of the target. The incorporation of no biotin was used as another negative control. Scheme 2C and 2D were similar to 2A and 2B, respectively except the additional use of 11-mercaptoundecanoic acid (MUA) as small molecules to covalently bind onto the gold surface and serve as a bridge to covalently connect to the proteins. These systems are believed to provide more stable and robust bindings and detection since covalent interactions are involved in the reactions. Table 1 shows the optimized component concentrations and reaction conditions for each system. Initial incubation of the proteins for a long period (13 to 16 hours) is important to secure strong adsorption onto either the bare or MUA-immobilized gold substrate which is helpful to attain robust and stable detection. The use of MUA in the case of Systems C and D creates better interaction with the gold substrates since the thiol group of MUA covalently binds with the gold in contrast to just immobilizing the proteins on the gold substrate. EDC/NHS was used to activate the carboxylic end group of the MUA to covalently react with the amine groups of the BSA and/or streptavidin. Systems A and C show initial evidence of the formation of protein-polymer conjugates on the gold substrates which can be used for the visual detection of biotin in a later process. The presence and visibility of spots on the gold substrates indicate a successful surface-initiated polymerization as shown in Figure 2. Figure 2A, 2B, 2F, and 2G show the substrates from system A and C after polymerization. It is clear from the figure that the positive controls (A and F) gave visible polymer spots measuring around 50 to 70 nm while the negative control (no CTA) did not give any spots. The polymer was formed on top of the streptavidin because of the presence of CTA which regulates and controls RAFT polymerization. Figure 1 shows the quantitative polymer film thickness values for all model systems. The negative control still measured around 3 to 10 nm, which is due to the proteins that were initially immobilized or some nonspecific polymer and/or monomer adsorption on the surface. Nonetheless, systems A and F gave a 95% reproducibility and high signal-to-noise ratio (S/N) of 13 and 7, respectively. Systems B and D were used as model systems for the detection of biotin. It involves a sandwich system among BSA, biotin, and streptavidin and is based on antibody-antigen interaction. This approach represents the actual detection of peanut allergens with BSA acting as the primary antibody, biotin as the target, and streptavidin as the secondary antibody. The CTA that was coupled to streptavidin will allow the formation of polymer film for visual detection of the target. The molar ratio of BSA and biotin used is 1:10 which was pre-mixed for one hour before immobilizing onto the substrates. This pre-mixing process is important to give enough time for the biotin to bind to BSA non-covalently. The strong affinity of biotin to streptavidin connects the two proteins on the surface while sandwiching the target biotin. Here, the theoretical amount of biotin (2.45 x 10-6 mmol) used was about 10 times in excess with respect to the amount of streptavidin (5.66 x 10-8 mmol) in the system since the washing step could affect the interaction of BSA and biotin. The formation of polymer film on top of streptavidin on the gold surface indicates a successful detection of biotin which are shown in Figures 2C and 2H as visible spots measuring around 60 to 70 nm. Without biotin, there was no interaction between BSA and streptavidin, ending up with the streptavidin being washed up during the washing step. As a result, no spots were obtained on the gold substrates as shown in Figures 2E and 2J. A high S/N of between 8 to 14 and 95% reproducibility were also obtained in these systems, which is indicative of the reliability and robustness of the process for the detection. The surface topography of the polymer film on the gold substrate was also examined using atomic force microscopy (AFM) as shown in Figure 3. The three-dimensional AFM images of the polymer film from model systems C and D after a 5-h RAFT polymerization process showed a rough surface with polymer islands distributed across it. The strong light scattering brought by these polymer islands caused the polymer spots to be visible and be directly observed on the surface.
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