Objective
The objective of this project is the development of a CKM system which will monitor BOHB concentration of normal (20 – 270µmol/L), threshold (1.2 mmol/L), to DKA level (> 3mmol/L), continuously, real-time, and stable for 2 weeks, which is composed of microsensor with novel BOHB sensing molecule, the BOHB binding protein (BBP), based electrochemical principle. The CKM hardware will be designed, fabricated, and tested by combining a miniaturized sensing electrode equipped with Bluetooth Low Energy (BLE) wireless transmission system and Low Power Potentiostat (LPP) suitable for in vivo monitoring of BOHB in ISF. Based on the achievement of this project, a CGM/CKM system will be developed, which will realize the alert system for hyperketonemia prior to the onset of ketoacidosis by predicting future potential DKA level, acknowledging the high sensitivity of the CKM system employing BBP based detection principle and real-time continuous monitoring of BOHB.
Project Deliverables:
1) Methods/Principle to electrochemically sense BOHB with BPP
2) Engineered BBP suitable for BOHB sensing
3) Electrochemical sensors for BOHB using BBP
4) Microelectrode suitable for transdermal BBP-based sensing
5) BLE wireless transmission system equipped with LPP monitoring system.
6) Continuous operation/monitoring protocols for continuous BOHB monitoring
Background Rationale
The current biggest challenge to realize CKM system, is the limited availability of molecular recognition molecules for BOHB monitoring. To achieve CKM with extended lifetime and accuracy, new molecular recognition molecules for BOHB, that can operate free of soluble cofactors, are necessary.
Current available principle for BOHB monitoring is limited to the use of the enzyme, 3-hydroxybutyrate dehydrogenase (BOHBDh), and this is the only enzyme discovered in the nature, which catalyzes the reaction of BOHB, and being utilized for either spectroscopic or electrochemical principle based enzyme analyses. Unlike glucose oxidase, the versatile enzyme utilized for the continuous glucose monitoring system, BOHBDh is suffering from the complexity in the redox-chemistry of NAD(H), as well as the requirement of the presence of NAD+ in the reaction mixture have been the technological limitation to construct the continuous monitoring system employing this enzyme.
In this project, we propose to develop a binding protein-based electrochemical micro-sensor and its application for CKM system. To break through the major technology limitation in the availabilities in BOHB sensing molecules, we introduce a novel engineered BOHB binding protein, which specifically and reversibly recognizes and binds with BOHB, suitable for continuous monitoring.
PI Sode’s research group has reported several biosensing technologies using PBPs, represented in the glucose binding protein based glucose monitoring, and glycated-amino acid binding protein based HbA1c sensing. The PI’s group has also reported on the genome-wide screening of PBPs for the unmet target sensing, engineering of PBPs to modify their substrate specificity, binding constant to be adjusted for the physiological concentration of the targets, and introduction of fluorescence probe modification, as well as their recombinant production/preparation. Sode’s group recently reported on the electrochemical continuous monitoring of L-glutamine using L-glutamine-binding protein. The achievement demonstrated the feasibility and superiority of PBP based electrochemical sensors for continuous monitoring system, which addresses the future development of an electrochemical biosensor for in situ, real-time, continuous monitoring of various metabolites based on PBPs.
Co-PI, Dr. Daniele with his collaborative team have reported wearable systems for minimally-invasive biosensing, point-of-care sensing, and continuous biochemical assays on dermal biofluids. This includes measurement of glucose, lactate, pH, and skin temperature via screen-printed sensors. They have demonstrated an electronic system in wristwatch form factors for wireless continuous electrochemical sensing of sweat. These systems include multiplexed potentiostats for multisensor operation form three discrete electrochemical cells. They have also developed a bandage size electrochemical sensing system with four flexible sensors screen-printed onto wound dressings for uric acid based wound healing monitoring. In addition, collaborative efforts have generated the necessary design rules and methods for engineering flexible electronic and optical devices, multi-layer conformal circuit boards, and integrated multimodal systems, by optimizing the trace geometries, circuit board materials, and device processing to achieve robust flexibility without loss of performance.
Co-PI, Dr. Tsugawa at Tokyo University of Agriculture and Technology, is the expert in the biosensor development as well as in the designing biomolecule for biosensors. Her research group has also varieties of experiences in the preparation of recombinant proteins for biosensors.
Sode’s group has been searching and developing novel biosensing molecules for BOHB, a BOHB binding proteins (BBPs), which is the ideal biosensing molecule for the development of the electrochemical sensor for continuous monitoring of BOHB. The recombinantly produced putative BBP was shown to bind BOHB and change its conformation dependent on the concentration of BOHB which was confirmed by the intrinsic fluorescence. Therefore, with the combination of our previous achievement in the development of electrochemical sensor with PBP, together with the technology development necessary to construct microelectrodes and electronics by Dr. Daniele, an innovative CKM sensing system will be developed.
Description of Project
Diabetic ketoacidosis (DKA) is a life-threatening complication which is characteristic in individual with insulin deficiency. The American Diabetes Association advises that blood ketone testing methods to quantify, D-β-hydroxybutyrate (abbreviated BOHB) would be desirable for diagnosis and monitoring of ketoacidosis. DKA occurs predominantly in Type 1 diabetic patients, but is also observed in Type 2 diabetes or gestational diabetes patients, and also serious DKA have been reported in type 2 diabetes patients treated with SGLT-2 inhibitors. Thus, ketone sensing technologies are receiving greater attention to prevent or improve outcomes of DKA. Especially, the precise dynamic information of blood ketone concentration and, “ketone variability” will be expected to manage and prevent DKA in the diabetic patients, which will be achieved by the development of continuous ketone monitoring (CKM) system.
Current available principle for BOHB monitoring is limited to the use of the enzyme, 3-hydroxybutyrate dehydrogenase (BOHBDh), and this is the only enzyme discovered in the nature, which catalyzes the reaction of BOHB, and being utilized for either spectroscopic or electrochemical principle based enzyme analyses. However, the principle of the measurement is not suitable for the continuous monitoring system, like the ones achieved for glucose monitoring. Unlike glucose oxidase/dehyrdrogenase which harbor a tightly bound cofactor (FAD), which is necessary molecule for the enzyme reaction, BOHBDh does not bind with the cofactor, NAD+, and must be added to the solution. The requirements to retain unstable, small, and soluble cofactor in the sensor, limit the technology development of the CKM system.
In this project, we propose to develop a binding protein-based electrochemical micro-sensor and its application for CKM system. To break through the major technology limitation in the availabilities in BOHB sensing molecules, we introduce a novel engineered BOHB binding protein, which specifically and reversibly recognizes and binds with BOHB, suitable for continuous monitoring. With the combination of the versatile and accurate electrochemical sensing principle, similar with the ones employed in CGM system, and microelectrode equipped with low-power requiring wireless transmission system and controller, we realize the in vivo, real-time continuous monitoring of BOHB in the interstitial fluid (ISF).
Anticipated Outcome
At the end of this project, a CKM system equipped with BLE wireless transmission system combined with LPP monitoring system will be developed. We will develop a miniaturized electrode, which will be fit to the inserters of sensor for CGMs, which are developed, approved by FDA, and commercialized by several medical device companies (e.g. Dexcom Inc.). Therefore, soon after we finish our animal experiments, we will plan to prepare for the clinical trial using devices and sensing platform derived from expected industrial partners’ CGM systems. We hope, we will be able to test our CKM system together with future industrial partner(s), within 2 years after the proposed project will finish, to develop CGM/CKM system which will realize the alert system for hyperketonemia prior to the onset of ketoacidosis by predicting future potential DKA level, acknowledging the high sensitivity of the CKM system employing BBP based detection principle and real-time continuous monitoring of BOHB.
One of the advantages of this proposed CKM system is the versatility of the sensing molecule, BBP, itself; not only electrochemical principle but BBP can be used for fluorescence monitoring system. By modifying with appropriate fluorophores, BBP conformation change based BOHB monitoring is promising, when fluorescence sensing platform is combined with this detection principle. Namely, if industries which are interested in, or have developed fluorescence based CGM systems (e.g. Eversense, Senseonics), our principle is readily available to develop long term implantable CKM system together with CGM system. Therefore, in the future, the development of the fluorescence based implantable CKM system together with appropriate industrial partners is an option.
Relevance to T1D
Diabetic ketoacidosis (DKA) is a life-threatening complication which is characteristic in individual with insulin deficiency. It occurs predominantly in Type 1 diabetic patients, but is also observed in Type 2 diabetes or gestational diabetes patients. DKA is resulted from the shortage of insulin and increased regulatory hormones like glucagon and cortisol, which leads to hyperglycemia and accumulation of ketones bodies in blood; R-3-hydroxybutyrate (or D-3-hydroxybutyrate, D-β-hydroxybutyrate, 3-D-hydroxybutyrate, abbreviated BOHB in this proposal) and acetoacetate, which is then broken down into acetone. The American Diabetes Association advises that blood ketone testing methods to quantify BOHB would be desirable for diagnosis and monitoring of ketoacidosis. Ketoacidosis occurs when ketone bodies are produced faster than metabolic break down. Euglycemic DKA has been identified as a potential morbidity in diabetic patients who take sodium-glucose co-transporter 2 (SGLT-2) inhibitor. SGLT-2 inhibitors block SGLT-2, which absorbs glucose from urine and returns it to the bloodstream as the blood is filtered in the kidneys. By blocking SGLT-2, these medicines cause more glucose to be eliminated in the urine, lowering the blood glucose level with an insulin independent mechanism. Patients treated with SGLT-2 inhibitors may be at higher risk for unrecognized DKA due to increased urinary glucose excretion (in the range of 50 to 100 g/day), resulting in lower blood glucose levels than would be expected in the onset of DKA. In clinical practice, rare but serious DKA have been reported in type 2 diabetes patients treated with SGLT-2 inhibitors and more commonly (absolute risk increase ~10% per year in type 1 diabetes. Thus, ketone sensing technologies are receiving greater attention to prevent or improve outcomes of DKA.
In DKA, BOHB is the predominant metabolite (BOHB 78%, acetoacetate 20%, acetone 2%). At present, the measurement of ketone bodies is performed using blood or urine as the sample. For the measurement of BOHB, blood or interstitial fluid (ISF) are considered better testing matrix, because urine does not equate to the plasma ketone concentration.
A commonly used rule for interpreting blood ketone levels is that normal levels of BOHB can be defined as <0.6 mM, ketosis can be defined as levels of 0.6-1.5 mM, hyperketonemia can be defined as levels of 1.5-3.0 mM, and ketoacidosis can be defined as levels in excess of 3.0 mM. However, current commercially available disposable single use sensor strips for blood ketone levels can only offer a snapshot that reflects ketonemia during a specific point in time.
Our goal of this project is to develop a CKM system which will monitor BOHB concentration 24/7, continuously, real-time, for a period of two weeks. Based on the achievement of this project, a CGM/CKM system will be developed, which will realize the alert system for hyperketonemia prior to the onset of ketoacidosis by predicting future potential DKA level, acknowledging the high sensitivity of the CKM system employing BBP based detection principle and real-time continuous monitoring of BOHB.
Based on thus developed CKM system, precise dynamic information of blood ketone concentration and, “ketone variability” will be expected to manage and prevent DKA in the diabetic patients.