This article demonstrates the sensitive detection and quanti

This article demonstrates the sensitive detection and quantification of C-reactive protein (CRP) using the designed MCFOB and an adaptive sandwich immunoassay [8,18,19]. The normal level of CRP in blood serum is 640ng/ml, but tends to slightly increase with age [13]. High concentrations of CRP are associated with an inflammation response. To simulate the serum sample, CRP is mixed with an interfering protein (bovine serum albumin) at a relatively high concentration. Furthermore, to demonstrate that the signal is generated only by EW, a real-time measurement using avidin and biotin-4-fluorescein is performed. Finally, to prove the chosen signal collection method is the most sensitive one, we compare the Fig. 1-a and -d geometries by detecting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the designed MCFOB.
Materials and methods A fused silica capillary with an outer diameter (O.D.) of 615μm and an inner diameter (I.D.) of 536μm was purchased from Polymicro Technologies (Phoenix, AZ, USA). (3-mercaptopropyl)-trimethoxy-silane (MPTMS) and trichloro(1 H,1 H,2 H,2 H-perfluorooctyl)silane (TCS) were purchased from Sigma-Aldrich (St. Louis, MO). Phosphate buffered saline (PBS), ethanol, n-(γ-maleimidobutyryloxy)succinimide ester (GMBS), and Tween-20 were purchased from Thermo Fisher Scientific (Pittsburgh, PA). Avidin, Alexa Fluor 647 and Alexa Fluor 488 antibody labeling kit were purchased from Invitrogen (Carlsbad, CA). Immunoassay grade bovine serum albumin (BSA) was purchased from MP Biomedicals (Santa Ana, CA). Human GAPDH, anti-GAPDH, biotinylated anti-GAPDH, recombinant human CRP, biotinylated anti-CRP antibody, and unconjugated anti-CRP antibody were purchased from R&D Systems (Minneapolis, MN). Hydrogen peroxide and sulfuric erk inhibitors were purchased from Avantor Performance Materials (Center Valley, PA). Sylgard 184 silicon elastomer kit was purchased from Dow Corning (Midland, MI). Carbonate paste BQ225 was purchased from DuPont (Wilmington, DE).
Results and discussion
Conclusion
Acknowledgments This work is supported by National Institute of General Medical Sciences (NIGMS) (R21GM103535). The author thanks Kirsten Jackson for the help in revising of the manuscript, and Dr. Xiangjun Zheng for the instruction on laser micromachining.
Introduction Biorecognition of proteins with molecular imprints (MI) attracts broad interests of applications in biodetection, biopurification and bioseparation. However, it is still a serious challenge due to the complexity of protein molecules [24]. Conventional approaches established for imprinting small molecules can\'t be directly applied to protein regarding the choices of monomers selection, temperature, pH, solvent, and ionic strength for polymer preparation. The massive amount residues on the protein surface can orchestrate a multitude of interactions with environmental molecules by hydrophobicity, electrical attraction, polarity, hydrogen bond, and van der Waals force. Traditionally, functional monomers (fMer) are used to pre-complex with the template based on the complementary interactions between two molecules. Then, the complex is fixed in the scaffold of polymerized cross-linking monomers [22]. The special requirements to design protein imprint reside in at least two folds: first, a fMer screening by computational chemoinformatics analysis is necessitated, yet the validated guideline and strategy are still unavailable. In line of such effort, GOLD package was used to study the docking of 7 fMers on human serum albumin [11]. It derived the characteristics of the fMer binding, as well as their probable interruption of the protein structures. Second, a complexation between multiple fMers and a protein could help to imprint recognition sites in different aspects of the protein molecule. In a pilot research, the statistical copolymerization of simple binding monomers in defined stoichiometric ratios had shown improved selectivity of protein imprints [10]. But as the number of functional monomer species increases, it becomes difficult to satisfy the reaction conditions simultaneously in a single polymerization system. To solve the problem, we should first have the binding details of the fMers for a given protein template, which could allow more freedom to choose compatible fMers to produce multiple recognition features in the imprint.