Fluctuations in the systemic concentration levels of metabolites, nutritionally relevant peptide hormones, protein biomarkers, and therapeutic materials provide a wealth of information that can be used to inform real-time clinical intervention. Thus, therapeutic outcomes for many disease states could be improved through the implementation of continuous monitoring systems. The most well-represented example of in vivo continuous monitoring of a small-molecule metabolite is the continuous glucose monitors used extensively in diabetes management. Unfortunately, to date, there is yet to be a marketed product that meets the engineering challenges or regulatory requirements for continuous sensing of peptides or proteins. A critical limitation of realizing this type of sensing is the limited availability of affinity-type biosensing elements, such as aptamers or antibodies. These molecules, while highly specific, have dissociation constants in the nano–picomolar range, which prevents reversibility between the biosensing element and analyte. In this review, several key challenges regarding the use of affinity-type biosensing elements to measure the concentration of peptides/proteins continuously in vivo are discussed. We discuss several examples of research groups working to overcome these limitations through specific engineering of biosensing elements, or by modulating the binding interaction itself using external energy. We then turn the discussion to insulin, a crucial therapeutic peptide for diabetes with the potential to enhance patient outcomes via continuous monitoring in vivo. This serves as a case study to explain why protein/peptide sensors currently suffer from translation. Finally, we summarize the current literature for insulin detection and discuss general translation toward in vivo continuous sensing of peptide/protein analytes.

Aptamer-based electrochemical sensors have gained attention in the context of developing a diagnostic biomarker detection method because of their rapid response, miniaturization ability, stability, and design flexibility. In such detection systems, enzymes are often used as labels to amplify the electrochemical signal. We have focused on glucose dehydrogenase (GDH) as a labeling enzyme for electrochemical detection owing to its high enzymatic activity, availability, and well-established electrochemical principle and platform. However, it is difficult and laborious to obtain one to one labeling of a GDH-aptamer complex with conventional chemical conjugation methods. In this study, we used GDH that was genetically fused to a DNA binding protein, i.e., zinc finger protein (ZF). Fused GDH can be attached to an aptamer spontaneously and site specifically in a buffer by exploiting the sequence-specific binding ability of ZF. Using such a fusion protein, we labeled a vascular endothelial growth factor (VEGF)-binding aptamer with GDH and detected the target electrochemically. As a result, upon the addition of glucose, the GDH labeled on the aptamer generated an amperometric signal, and the current response increased dependent on the VEGF concentration. Eventually, the developed electrochemical sensor proved to detect VEGF levels as low as 105 pM, thereby successfully demonstrating the concept of using ZF-fused GDH to enzymatically label aptamers.

Single-chain fragment variables (scFvs), composed of variable heavy and light chains joined together by a peptide linker, can be produced using a cost-effective bacterial expression system, making them promising candidates for pharmaceutical applications. However, a versatile method for monitoring recombinant-protein production has not yet been developed. Herein, we report a novel anti-scFv aptamer-based biosensing system with high specificity and versatility. First, anti-scFv aptamers were screened using the competitive systematic evolution of ligands by exponential enrichment, focusing on a unique scFv-specific peptide linker. We selected two aptamers, P1-12 and P2-63, with K_D = 2.1 μM or K_D = 1.6 μM toward anti-human epidermal growth factor receptor (EGFR) scFv, respectively. These two aptamers can selectively bind to scFv but not to anti-EGFR Fv. Furthermore, the selected aptamers recognized various scFvs with different CDRs, such as anti-4-1BB and anti-hemoglobin scFv, indicating that they recognized a unique peptide linker region. An electrochemical sensor for anti-EGFR scFv was developed using anti-scFv aptamers based on square wave voltammetry. Thus, the constructed sensor could monitor anti-EGFR scFv concentrations in the range of 10-500 nM in a diluted medium for bacterial cultivation, which covered the expected concentration range for the recombinant production of scFvs. These achievements promise the realization of continuous monitoring sensors for pharmaceutical scFv, which will enable the real-time and versatile monitoring of large-scale scFv production.

One challenge in the continuous monitoring of insulin lies with the unavailability of a high-affinity biological recognition element (BRE) with kinetic parameters sufficient to track fluctuating concentrations of insulin in vivo. An approach to overcome this limitation is to engineer a high-affinity BRE to selectively modulate its binding kinetics in response to an external signal. Herein, we design and evaluate changes in the pH-dependent binding kinetics of an anti-insulin single chain variable fragment (scFv) we previously employed in a point-of-care insulin sensor. We predicted the scFv structure in complex with human insulin and selected scFv residues directly involved in insulin binding for histidine substitution. We identify one mutation, T32H, that improves the pH-sensitivity of the wild-type (WT); the K_D of the T32H mutant is calculated to be 145.5 ± 83.1 nM at pH 7.4 and 17.4 ± 5.1 nM at pH 6.0 – an average of an 8.4× difference between the two conditions and a 3.8× increase in pH-sensitivity from the WT. We design a bio-layer interferometry (BLI) assay to interrogate the improved pH-sensitivity of the T32H mutant in tracking fluctuating insulin concentrations in dynamic pH conditions and find that improved pH-sensitivity can be leveraged to improve biosensor regeneration. These results suggest the potential for pH-sensitive antibodies to improve the development of in vivo continuous monitoring systems.