Developing point-of-care (POC) sensors for rapid blood analysis is crucial for precision medicine. Blood contains a wealth of information about an individual's physiological state, including nutritional status, metabolites, disease markers, therapeutic response, and environmental influences. However, the opacity, complexity, and cellular components of blood pose significant challenges to sensing. Adapting existing glucometers, which are widely available and inexpensive, presents a promising avenue for creating diverse POC sensors. Previous work has demonstrated the modulation of glucose oxidation signals using engineered glucose dehydrogenase (GDH), incorporating analyte-specific recognition proteins to detect various compounds. However, challenges remain in developing effective electrochemical signal transmission and integrating these sensors into self-contained POC devices. This study focuses on developing a glucometer-based allosteric sensor (GBAS) to electrochemically detect specific biomarkers in blood, meeting the miniaturization and signal amplification needs of POC applications. Given the importance of frequent monitoring in preventing drug resistance or cancer recurrence, the researchers chose to develop a sensor for 4-hydroxytamoxifen (4-HT), a key metabolite of tamoxifen used in hormone receptor-positive breast cancer treatment. The sensor design utilizes the electrical current generated from enzymatic glucose oxidation to report the presence of 4-HT via engineered allostery. An electrochemical algorithm will decode the 4-HT signal, compensating for variations in glucose levels. Furthermore, the sensor will be self-powered using glucose, eliminating the need for external batteries, and amplified with an organic electrochemical transistor (OECT) for improved sensitivity.

The existing literature highlights the significance of point-of-care diagnostics for improving healthcare access and efficiency. The use of glucometers as adaptable platforms for various clinical analyses has been extensively explored, with researchers successfully modifying glucose dehydrogenase (GDH) to detect different analytes like rapamycin, tacrolimus, amylase, and cyclosporine A by incorporating analyte-specific recognition proteins. However, these studies primarily focus on the development of the sensing element, and the integration of these elements into functional, miniaturized, and self-powered POC devices remains a challenge. Studies on allosteric protein engineering have demonstrated the potential of manipulating protein structure to create switches that regulate diverse outputs, including fluorescence, cell growth, etc. However, predicting insertion sites that maintain protein stability and efficient signal transduction remains a significant hurdle. The use of electrochemical transistors, specifically organic electrochemical transistors (OECTs), for signal amplification in biosensing applications has also shown promise, offering a pathway to improve sensitivity and reduce the impact of noise.

The researchers developed a glucometer-based allosteric sensor (GBAS) by genetically fusing the ligand-binding domain (LBD) of the estrogen receptor alpha (ERα) with pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH). A comprehensive library of LBD-GDH insertion variants was created using the targeted domain insertion library technique. Deep sequencing analysis provided comprehensive insertion coverage. The library was screened using a colorimetric assay based on DCPIP reduction, identifying insertion sites that maintained GDH activity and exhibited 4-HT-regulatable activity. Structural analysis of GDH revealed that allosteric insertion sites are concentrated on flexible loops and the dimeric interface, emphasizing the importance of conformational flexibility for allosteric signaling. GDH-5E, a promising variant, was further engineered to enhance allosteric modulation. To enable current flow to an electrode, GDH-5E was immobilized with a redox polymer (Fc-LPEI). The resulting hydrogel facilitated electron transfer from PQQ to the electrode, preserving the 4-HT-mediated repression of GDH-5E activity. Amperometric measurements demonstrated the ability of this system to report 4-HT signals in a manner analogous to glucose sensing. To create a sample-to-result system, an electrochemical algorithm was developed. This algorithm normalizes the current from GDH-5E to the current from wild-type GDH to mitigate the influence of glucose concentration. The ratio of the two currents (iGDH-5E+/iGDH) served as the indicator for 4-HT presence. A dual-electrode setup was used to obtain both currents simultaneously. For self-powering, the researchers integrated GDH-5E+ into an enzymatic fuel cell (EFC), using glucose oxidation at the anode and laccase-catalyzed oxygen reduction at the cathode. Finally, to amplify the signal, the self-powered sensor was coupled with an OECT, utilizing the EFC's output as the gate voltage for the OECT, resulting in milliampere-level signals. Detailed methods are provided for all aspects, including protein expression, purification, characterization, electrode fabrication, electrochemical measurements, EFC construction, and OECT integration. The use of human blood adhered to strict ethical guidelines and protocols.

The study successfully created a library of GDH variants with the ERα LBD inserted at various positions. Deep sequencing revealed comprehensive insertion coverage. Screening this library identified 71 4-HT-regulatable variants, demonstrating the robustness of GDH as a scaffold for domain insertion. Structural analysis indicated that allosteric sites are concentrated on flexible loops and the dimeric interface of GDH, suggesting the importance of conformational flexibility in allosteric signaling. The engineered GDH-5E variant exhibited a significant decrease in glucose oxidation rate in the presence of 4-HT. Immobilization of GDH-5E with Fc-LPEI successfully wired the enzyme to an electrode, enabling amperometric detection of 4-HT. The electrochemical algorithm, based on the ratio of currents from GDH-5E+ and wild-type GDH, effectively mitigated the interference from varying glucose levels and provided a binary (yes/no) determination of 4-HT presence. The self-powered EFC sensor generated power from glucose oxidation, with 4-HT presence causing a reduction in current density. Coupling the self-powered sensor with an OECT amplified the 4-HT signal significantly, producing milliampere-level signals. This amplification occurred by observing a reduced rate of change in source-drain current (*dIsd/dt*) in the presence of 4-HT. These results successfully demonstrate a proof-of-concept device that demonstrates the feasibility of a miniaturized, self-powered, and sensitive 4-HT biosensor.

This study's findings address the critical need for simple and reliable POC sensors for therapeutic drug monitoring. The development of the GBAS successfully addresses several limitations of existing approaches. The use of an electrochemical algorithm to decode the 4-HT signal from the glucose signal effectively reduces interference from fluctuating glucose concentrations, providing a more accurate and robust sensing platform. The self-powered nature of the sensor eliminates the need for bulky and expensive external power supplies, enhancing the portability and usability of the device. The integration of an OECT significantly amplifies the signal, improving sensitivity and enabling miniaturization. The broad applicability of this approach is highlighted by the sensor's selectivity for 4-HT over other endocrine therapeutics. The successful demonstration of the GBAS opens exciting possibilities for real-time monitoring of therapeutic drug levels in clinical settings. The simplicity, low cost, and high sensitivity of the sensor make it suitable for widespread use.

This study presents a novel approach to developing a POC biosensor for 4-HT using an interdisciplinary approach that integrates protein engineering, electrochemical sensing, and electronic circuit design. The successful creation of a self-powered sensor with OECT amplification demonstrates the feasibility of a portable, sensitive, and cost-effective device for therapeutic drug monitoring. While the current prototype requires further development for continuous real-time monitoring, potential advancements such as using multiple EFCs in series or employing magnetic field powering could enable the creation of wearable or implantable versions of the sensor. Future work will focus on optimizing the sensor's performance, exploring its applicability to other therapeutic agents, and developing a fully integrated POC device.

The current prototype EFC is insufficient for real-time, in situ sensing and lacks the power to drive conventional transmitters. Future work is needed to address this limitation, possibly through using multiple EFCs in series or alternative power sources. The study used a single-donor human blood sample, potentially limiting the generalizability of the findings. Further testing with a larger and more diverse sample population is needed to confirm the sensor's performance across various physiological conditions. While the OECT amplification was successful, further optimization of the OECT design and integration with the EFC could lead to even greater signal enhancement.