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    Biomedical Imaging and Instrumentation

    The Effect of Protein G Binding with CKMB Antibody on TESPSA Modified PC-TIR Sensor

    (E-190) The Effect of Protein G Binding with CKMB Antibody on TESPSA Modified PC-TIR Sensor

    Saturday, October 14, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row E - Poster # 190

      Presenting Author(s)

    • MA

      Maisha Ahmed (she/her/hers)

      Procedure Technician
      University of Texas San Antonio BMES
      San Antonio, Texas, United States

      • Co-Author(s)

    • LT

      Luis Trabucco

      PhD Student
      University of Texas San Antonio, Texas, United States

      • Last Author(s)

    • JY

      Jingyong Ye

      Professor
      University of Texas San Antonio, United States

      • Co-Author(s)

    • VP

      Vidhi Patel

      Student
      University of Texas San Antonio, United States

    • JS

      Jonathan Shaw

      Grad Student
      University of Texas San Antonio, United States

    Introduction::

    Myocardial cells that are damaged release specific biomarkers, like creatine kinase-MB (CKMB), into the bloodstream. Early detection of these cardiac biomarkers is crucial for identifying myocardial infarctions and screening for potential drug cardiotoxicity. In this research, we utilized a compact, independent biosensing system containing a Photonic-Crystal structure in a Total-Internal-Reflection (PC-TIR) configuration for label-free detection of cardiac biomarkers. This system operates by monitoring subtle shifts in the resonance of the sensor to track analyte binding events on the nanoscale. A microfluidic channel system is attached atop the surface of the sensor for analysis of binding coefficients and to promote rapid binding of analytes through turbulent flow through the channels. For this application, functionalization of the PC-TIR sensor was achieved by addition of Triethoxysilylpropyl Succinic Anhydride (TESPSA) to the sensing surface. TESPSA was subsequently activated via hydrolyzed ring-opening with Recombinant Protein G (RPG) to aid in controlling antibody orientation for the label-free bioassay capable of rapid detection of CKMB.



    Materials and Methods:: The BK7 sensors’ OH groups were activated through plasma cleaning. Subsequently, TESPSA was introduced to activate an amine terminated surface. The sensor was then incubated overnight in 10% TESPSA in anhydrous toluene, carefully sealed and purged with N2 to prevent hydrolysis of TESPSA. The sensor was then sonicated twice in anhydrous methanol for 15 minutes to remove any unbound, or loosely bound TESPSA. Afterward, the sensor was annealed by baked at 140°C for 90 minutes to prevent optical drift during analysis. Using a microfluidic system, RPG was injected at a rate of 27 µL/min. to further functionalize TESPSA. The microfluidic system is a 3D-printed mold made of PDMS, creating 6 channels with 1 mm diameter and 100 µm in height. Following a 30-minute interval, antibody was introduced into the system. The quantity of immobilized antibodies under various conditions was ascertained through continuous monitoring of the sensor's resonance shift.

    Results, Conclusions, and Discussions::

    Scheme 1 depicts the salinization and ring-opening process of TESPSA on the PC-TIR sensor. For functionalization, Protein G was flowed over the TESPSA coated sensor in an aqueous solution, inducing hydrolysis, facilitating the ring-opening for Protein G binding. A homemade MATLAB program was used to process and analyze the data in real-time. Fig. 2 shows the binding affinity of Protein G to the TESPSA modified surface. Fig. 3 shows that channels Protein G1 and Protein G2 has a higher relative pixel shift compared to the CKMB Antibody alone exhibits lower relative pixel shift. This study outlines a robust label-free method to functionalize our PC-TIR biosensor. The results obtained from the biosensor and processed through our homemade MATLAB program reveal the high binding affinity of both Protein G and CKMB Antibody. This enables further enhanced immobilization of antigen molecules, facilitating sensitive label-free bioassays. Further application of the sensor could be immobilization and characterization of cardiac biomarker such as cTnI and CKMB antigen.



    Acknowledgements (Optional): :

    The authors are grateful for the grant support R01GM126571 by the National Institutes of Health.



    References (Optional): :