A Glucose Biosensor Based on Phosphorescence Lifetime Sensing and a Thermoresponsive Membrane.

The adoption of existing continuous glucose monitors (CGMs) is limited by user burden. Herein, a design for a glucose biosensor with the potential for subcutaneous implantation, without the need for a transcutaneous probe or affixed transmitter, is presented. The design is based on the combination of an enzyme-driven phosphorescence lifetime-based glucose-sensing assay and a thermoresponsive membrane anticipated to reduce biofouling. The metalloporphyrin, Pd meso-tetra(sulfophenyl)-tetrabenzoporphyrin ([PdPh4 (SO3 Na)4 TBP]3 , HULK) as well as glucose oxidase (GOx) are successfully incorporated into the UV-cured double network (DN) membranes by leveraging electrostatic interactions and covalent conjugation, respectively. The oxygen-sensitive metalloporphyrin is incorporated at different levels within the DN membranes. These HULK-containing membranes retain the desired thermosensitivity, as well as glucose diffusivity and primary optical properties of the metalloporphyrin. After subsequently modifying the membranes with GOx, glucose-sensing experiments reveal that membranes prepared with the lowest GOx level exhibit the expected increase in phosphorescent lifetime for glucose concentrations up to 200 mg dL-1 . For membranes prepared with relatively higher GOx, oxygen-limited behavior is considered the source of diminished sensitivity at higher glucose levels. This proof-of-concept study demonstrates the promising potential of a biosensor design integrating a specific optical biosensing chemistry into a thermoresponsive hydrogel membrane.

An Optical Urate Biosensor Based on Urate Oxidase and Long-Lifetime Metalloporphyrins.

Gout is a condition that affects over 8 million Americans. This condition is characterized by severe pain, and in more advanced cases, bone erosion and joint destruction. This study explores the fabrication and characterization of an optical, enzymatic urate biosensor for gout management, and the optimization of the biosensor response through the tuning of hydrogel matrix properties. Sensors were fabricated through the co-immobilization of oxygen-quenched phosphorescent probes with an oxidoreductase within a biocompatible copolymer hydrogel matrix. Characterization of the spectral properties and hydrogel swelling was conducted, as well as evaluation of the response sensitivity and long-term stability of the urate biosensor. The findings indicate that increased acrylamide concentration improved the biosensor response by yielding an increased sensitivity and reduced lower limit of detection. However, the repeatability and stability tests highlighted some possible areas of improvement, with a consistent response drift observed during repeatability testing and a reduction in response seen after long-term storage tests. Overall, this study demonstrates the potential of an on-demand, patient-friendly gout management tool, while paving the way for a future multi-analyte biosensor based on this sensing platform.

Hydrogel Microdomain Encapsulation of Stable Functionalized Silver Nanoparticles for SERS pH and Urea Sensing.

Conceptual and commercial examples of implantable sensors have been limited to a relatively small number of target analytes, with a strong focus on glucose monitoring. Recently, surface-enhanced Raman spectroscopy (SERS) pH sensors were demonstrated to track acid-producing enzymatic reactions targeting specific analytes. We show here that SERS pH tracking in the basic regime is also possible, and can be used to monitor urea concentration. To accomplish this, we developed a hydrogel consisting of polyelectrolyte multilayer microcapsules containing a SERS-sensitive pH reporter (4-mercapopyridine capped silver nanoparticles modified with bovine serum albumin). This pH sensing material exhibited a sensitive Raman scattering response to a wide range of pH from 6.5-9.7. By incorporating urease into the hydrogel matrix, the new sensor was capable of distinguishing urea concentrations of 0, 0.1, 1, and 10 mM. We also found that bovine serum albumin (BSA) prevented severe aggregation of the nanoparticle-based pH sensor, which improved sensing range and sensitivity. Furthermore, BSA safeguarded the pH sensor during the encapsulation procedure. Together, the combination of materials represents a novel approach to enabling optical sensing of reactions that generate pH changes in the basic range.

Albuminated Glycoenzymes: Enzyme Stabilization through Orthogonal Attachment of a Single-Layered Protein Shell around a Central Glycoenzyme Core.

Here we demonstrate an approach to stabilize enzymes through the orthogonal covalent attachment of albumin on the single-enzyme level. Albuminated glycoenzymes (AGs) based upon glucose oxidase and catalase from Aspergillus niger were prepared in this manner. Gel filtration chromatography and dynamic light scattering support modification, with an increase in hydrodynamic radius of ca. 60% upon albumination. Both AGs demonstrate a marked resistance to aggregation during heating to 90 °C, but this effect is more profound in albuminated catalase. The functional characteristics of albuminated glucose oxidase vary considerably with exposure type. The AG's thermal inactivation is reduced more than 25 times compared to native glucose oxidase, and moderate stabilization is observed with one month storage at 37 °C. However, albumination has no effect on operational stability of glucose oxidase.

Sequential Thiol-Ene and Tetrazine Click Reactions for the Polymerization and Functionalization of Hydrogel Microparticles.

Click chemistry is a versatile tool for the synthesis and functionalization of polymeric biomaterials. Here, we describe a versatile new strategy for producing bioactive, protein-functionalized poly(ethylene glycol) (PEG) hydrogel microparticles that is based on sequential thiol-ene and tetrazine click reactions. Briefly, tetra-functional PEG-norbornene macromer and dithiothreitol (SH) cross-linker were combined at a 0.75:1 [SH]:[norbornene] ratio, emulsified in a continuous Dextran phase, and then photopolymerized to form PEG hydrogel microparticles that varied from 8 to 30 µm in diameter, depending on the PEG concentration used. Subsequently, tetrazine-functionalized protein was conjugated to unreacted norbornene groups in the PEG microparticles. Tetrazine-mediated protein tethering to the microparticles was first demonstrated using fluorescein-labeled ovalbumin as a model protein. Subsequently, bioactive protein tethering was demonstrated using alkaline phosphatase (ALP) and glucose oxidase (GOx). Enzyme activity assays demonstrated that both ALP and GOx maintained their bioactivity and imparted tunable bioactivity to the microparticles that depended on the amount of enzyme added. ALP-functionalized microparticles were also observed to initiate calcium phosphate mineralization in vitro when incubated with calcium glycerophosphate. Collectively, these results show that protein-functionalized hydrogel microparticles with tunable bioactive properties can be easily synthesized using sequential click chemistry reactions. This approach has potential for future applications in tissue engineering, drug delivery, and biosensing.

Prevalence of Pseudotumor in Patients After Metal-On-Metal Hip Arthroplasty Evaluated with Metal Ion Analysis and MARS-MRI.

The purpose of this study is to quantify the prevalence of pseudotumors in patients with well-functioning and painful metal-on-metal total hip arthroplasty, to characterize the pseudotumor with the use of MARS-MRI, and to assess the relationship between pseudotumors and metal ions. We retrospectively reviewed 102 single surgeon patients. The results showed that 68.6% developed pseudotumor with 60.9% of the asymptomatic group developing pseudotumor. The symptomatic group had a higher proportion of patients with elevated serum cobalt levels (P=0.035). There was no difference found with elevated metal ions and prevalence of pseudotumor, but elevated cobalt levels were associated with larger pseudotumor size (P=0.001). The available evidence indicated that most patients that develop pseudotumors are asymptomatic, and that elevated serum cobalt levels may be associated with symptoms and pseudotumor size.

Temperature Compensation of Oxygen Sensing Films Utilizing a Dynamic Dual Lifetime Calculation Technique.

With advances to chemical sensing, methods for compensation of errors introduced by interfering analytes are needed. In this work, a dual lifetime calculation technique was developed to enable simultaneous monitoring of two luminescence decays. Utilizing a windowed time-domain luminescence approach, the response of two luminophores is separated temporally. The ability of the dual dynamic rapid lifetime determination (DDRLD) approach to determine the response of two luminophores simultaneously was investigated through mathematical modeling and experimental testing. Modeling results indicated that lifetime predictions will be most accurate when the ratio of the lifetimes from each luminophore is at least three and the ratio of intensities is near unity. In vitro experiments were performed using a porphyrin that is sensitive to both oxygen and temperature, combined with a temperature-sensitive inorganic phosphor used for compensation of the porphyrin response. In static experiments, the dual measurements were found to be highly accurate when compared to single-luminophore measurements-statistically equivalent for the long lifetime emission and an average difference of 2% for the short lifetimes. Real-time testing with dynamic windowing was successful in demonstrating dual lifetime measurements and temperature compensation of the oxygen sensitive dye. When comparing the actual oxygen and temperature values with predictions made using a dual calibration approach, an overall difference of less than 1% was obtained. Thus, this method enables rapid, accurate extraction of multiple lifetimes without requiring computationally intense curve fitting, providing a significant advancement toward multi-analyte sensing and imaging techniques.

Comparison of custom to standard TKA instrumentation with computed tomography.

PURPOSE: There is conflicting evidence whether custom instrumentation for total knee arthroplasty (TKA) improves component position compared to standard instrumentation. Studies have relied on long-limb radiographs limited to two-dimensional (2D) analysis and subjected to rotational inaccuracy. We used postoperative computed tomography (CT) to evaluate preoperative three-dimensional templating and CI to facilitate accurate and efficient implantation of TKA femoral and tibial components. METHODS: We prospectively evaluated a single-surgeon cohort of 78 TKA patients (51 custom, 27 standard) with postoperative CT scans using 3D reconstruction and contour-matching technology to preoperative imaging. Component alignment was measured in coronal, sagittal and axial planes. RESULTS: Preoperative templating for custom instrumentation was 87 and 79 % accurate for femoral and tibial component size. All custom components were within 1 size except for the tibial component in one patient (2 sizes). Tourniquet time was 5 min longer for custom (30 min) than standard (25 min). In no case was custom instrumentation aborted in favour of standard instrumentation nor was original alignment of custom instrumentation required to be adjusted intraoperatively. There were more outliers greater than 2° from intended alignment with standard instrumentation than custom for both components in all three planes. Custom instrumentation was more accurate in component position for tibial coronal alignment (custom: 1.5° ± 1.2°; standard: 3° ± 1.9°; p = 0.0001) and both tibial (custom: 1.4° ± 1.1°; standard: 16.9° ± 6.8°; p < 0.0001) and femoral (custom: 1.2° ± 0.9°; standard: 3.1° ± 2.1°; p < 0.0001) rotational alignment, and was similar to standard instrumentation in other measurements. CONCLUSIONS: When evaluated with CT, custom instrumentation performs similar or better to standard instrumentation in component alignment and accurately templates component size. Tourniquet time was mildly increased for custom compared to standard.

Implementing a Teaching Rural Mobile Health Clinic: Challenges and Adaptations.

In 2022, Penn State College of Medicine launched the LION Mobile Clinic, a teaching mobile health clinic offering preventive health services in rural Snow Shoe, Pennsylvania. We outline four challenges the clinic team faced in implementation, along with adaptations made to tailor the model to Snow Shoe's needs and opportunities.

Long-Term Evaluation of Inserted Nanocomposite Hydrogel-Based Phosphorescent Oxygen Biosensors: Evolution of Local Tissue Oxygen Levels and Foreign Body Response.

Phosphorescence-based oxygen-sensing hydrogels are a promising platform technology for an upcoming generation of insertable biosensors that are smaller, softer, and potentially more biocompatible than earlier designs. However, much remains unknown about their long-term performance and biocompatibility in vivo. In this paper, we design and evaluate a range of hydrogel sensors that contain oxygen-sensitive phosphors stabilized by micro- and nanocarrier systems. These devices demonstrated consistently good performance and biocompatibility in young adult rats for over three months. This study thoroughly establishes the biocompatibility and long-term suitability of phosphorescence lifetime sensors in vivo, providing the groundwork for expansion of this platform technology into a family of small, unobtrusive biosensors for a range of clinically relevant metabolites.

NIR Luminescent Oxygen-Sensing Nanoparticles for Continuous Glucose and Lactate Monitoring.

A highly sensitive, biocompatible, and scalable phosphorescent oxygen sensor formulation is designed and evaluated for use in continuous metabolite sensors for biological systems. Ethyl cellulose (EC) and polystyrene (PS) nanoparticles (NPs) stabilized with Pluronic F68 (PF 68), Polydimethylsiloxane-b-polyethyleneglycol methyl ether (PDMS-PEG), sodium dodecylsulfate (SDS), and cetyltimethylammonium bromide (CTAB) were prepared and studied. The resulting NPs with eight different surfactant−polymer matrix combinations were evaluated for physical properties, oxygen sensitivity, effect of changes in dispersion matrix, and cytotoxicity. The EC NPs exhibited a narrower size distribution and 40% higher sensitivity than PS, with Stern−Volmer constants (Ksv) 0.041−0.052 µM−1 for EC, compared to 0.029−0.034 µM−1 for PS. Notably, ethyl cellulose NPs protected with PF68 were selected as the preferred formulation, as they were not cytotoxic towards 3T3 fibroblasts and exhibited a wide phosphorescence lifetime response of >211.1 µs over 258−0 µM and ~100 µs over 2.58−0 µM oxygen, with a limit of detection (LoD) of oxygen in aqueous phase of 0.0016 µM. The EC-PF68 NPs were then efficiently encapsulated in alginate microparticles along with glucose oxidase (GOx) and catalase (CAT) to form phosphorescent nanoparticles-in-microparticle (NIMs) glucose sensing microdomains. The fabricated glucose sensors showed a sensitivity of 0.40 µs dL mg−1 with a dynamic phosphorescence lifetime range of 46.6−197.1 µs over 0−150 mg dL−1 glucose, with a glucose LoD of 18.3 mg dL−1 and maximum distinguishable concentration of 111.1 mg dL−1. Similarly, lactate sensors were prepared with NIMs microdomains containing lactate oxidase (LOx) and found to have a detection range of 0−14 mg dL−1 with LoD of 1.8 mg dL−1 and maximum concentration of 13.7 mg dL−1 with lactate sensitivity of 10.7 µs dL mg−1. Owing to its versatility, the proposed NIMs-based design can be extended to a wide range of metabolites and different oxygen-sensing dyes with different excitation wavelengths based on specific application.

Glucose biosensors based on Michael addition crosslinked poly(ethylene glycol) hydrogels with chemo-optical sensing microdomains.

Continuous glucose monitoring (CGM) devices have the potential to lead to better disease management and improved outcomes in patients with diabetes. Chemo-optical glucose sensors offer a promising, accurate, long-term alternative to the current CGMs that require frequent calibration and replacement. Recently, we have proposed glucose sensor designs using phosphorescence lifetime-based measurement of chemo-optical glucose sensing microdomains embedded within alginate hydrogels. Due to the poor long-term stability of calcium-crosslinked alginate, we propose poly(ethylene glycol) (PEG) hydrogels synthesized via thiol-Michael addition chemistry as an alternative hydrogel carrier. The objective of this study was to evaluate the suitability of Michael addition crosslinked PEG hydrogels compared to calcium crosslinked alginate hydrogels for encapsulating glucose-sensing microdomains. PEG hydrogels crosslinked via thiol-vinyl sulfone addition achieved gelation in under 5 minutes, resulting in an even distribution of sensing microdomains. The shear storage modulus of the PEG hydrogels was tunable from 2.2 ± 0.1 kPa to 9.5 ± 1.8 kPa, which was comparable to the alginate hydrogels (10.5 ± 0.8 kPa), and the inclusion of microdomains did not significantly impact stiffness. The high water content of PEG hydrogels resulted in high glucose permeability that closely corresponded to the glucose permeability of alginate (D = 0.09 and 0.12 cm2 s-1, respectively; p = 0.47), but the PEG hydrogels exhibited superior stability. Both PEG and alginate-embedded sensors exhibited a sensing range up to ∼200 mg dL-1 glucose. The lower limits of detection (LOD) for PEG and alginate-based glucose sensors were 19.8 and 20.6 mg dL-1 with a difference of just 4.2% variation. The small difference between PEG and alginate embedded sensors indicates that their sensing properties are primarily determined by the glucose sensing microdomains rather than the hydrogel matrix. Overall, the results of this study indicate that Michael addition-crosslinked PEG hydrogels are a promising platform for encapsulation of chemo-optical glucose sensing microdomains.

Dynamic windowing algorithm for the fast and accurate determination of luminescence lifetimes.

An algorithm for the accurate calculation of luminescence lifetimes in near-real-time is described. The dynamic rapid lifetime determination (DRLD) method uses a window-summing technique and dynamically selects the appropriate window width for each lifetime decay such that a large range of lifetimes can be accurately calculated. The selection of window width is based on an optimal range of window-sum ratios. The algorithm was compared to alternative approaches for rapid lifetime determination as well as nonlinear least-squares (NLLS) fitting in both simulated and real experimental conditions. A palladium porphyrin was used as a model luminophore to quantitatively evaluate the algorithm in a dynamic situation, where oxygen concentration was modulated to induce a change in lifetime. Unlike other window-summing techniques, the new algorithm calculates lifetimes that are not significantly different than the slower, traditional NLLS. In addition, the computation time required to calculate the lifetime is 4 orders of magnitude less than NLLS and 2 orders less than other iterative methods. This advance will improve the accuracy of real-time measurements that must be made on samples that are expected to exhibit widely varying lifetimes, such as sensors and biosensors.

Geometry design for a fully insertable glucose biosensor with multimodal optical readout.

Significance: Insertable optical continuous glucose monitors (CGMs) with wearable readers are a strong option for monitoring individuals with diabetes. However, a fully insertable CGM requires a small form factor while still delivering sufficient signal to be read through tissue by an external device. Previous work has suggested that a multimodal repeating unit (barcode) approach may meet these requirements, but the biosensor geometry must be optimized to meet performance criteria. Aim: This work details in silico trials conducted to evaluate the geometry of a fully insertable multimodal optical biosensor with respect to both optical output and species diffusion in vivo. Approach: Monte Carlo modeling is used to evaluate the luminescent output of three presupposed biosensor designs based on size constraints for an injectable and logical placement of the bar code compartments. Specifically, the sensitivity of the luminescent output to displacement of the biosensor in the X and Y directions, overall size of the selected design, and size of an individual repeating unit are analyzed. Further, an experimentally validated multiphysics model is used to evaluate the diffusion and reaction of glucose and oxygen within the biosensor to estimate the occurrence of chemical crosstalk between the assay components. Results: A stacked cylinder multimodal biosensor 4.4 mm in length with repeating units 0.36 mm in length was found to yield a greater luminescent output than the current "barcode" biosensor design. In addition, it was found that a biosensor with enzymatic elements does not significantly deplete glucose locally and thus does not impact the diffusion profile of glucose in adjacent compartments containing nonenzymatic assays. Conclusions: Computational modeling was used to design the geometry of a multimodal, insertable, and optical CGM to ensure that the optical output and chemical diffusion profile are sufficient for this device to function in vivo.

Monte Carlo method for assessment of a multimodal insertable biosensor.

SIGNIFICANCE: Continuous glucose monitors (CGMs) are increasingly utilized as a way to provide healthcare to the over 10% of Americans that have diabetes. Fully insertable and optically transduced biosensors are poised to further improve CGMs by extending the device lifetime and reducing cost. However, optical modeling of light propagation in tissue is necessary to ascertain device performance. AIM: Monte Carlo modeling of photon transport through tissue was used to assess the luminescent output of a fully insertable glucose biosensor that uses a multimodal Förster resonance energy transfer competitive binding assay and a phosphorescence lifetime decay enzymatic assay. APPROACH: A Monte Carlo simulation framework of biosensor luminescence and tissue autofluorescence was built using MCmatlab. Simulations were first validated against previous research and then applied to predict the response of a biosensor in development. RESULTS: Our results suggest that a diode within the safety standards for light illumination on the skin, with far-red excitation, allows the luminescent biosensor to yield emission strong enough to be detectable by a common photodiode. CONCLUSIONS: The computational model showed that the expected fluorescent power output of a near-infrared light actuated barcode was five orders of magnitude greater than a visible spectrum excited counterpart biosensor.

A Computational Modeling and Simulation Workflow to Investigate the Impact of Patient-Specific and Device Factors on Hemodynamic Measurements from Non-Invasive Photoplethysmography.

Cardiovascular disease is the leading cause of death globally. To provide continuous monitoring of blood pressure (BP), a parameter which has shown to improve health outcomes when monitored closely, many groups are trying to measure blood pressure via noninvasive photoplethysmography (PPG). However, the PPG waveform is subject to variation as a function of patient-specific and device factors and thus a platform to enable the evaluation of these factors on the PPG waveform and subsequent hemodynamic parameter prediction would enable device development. Here, we present a computational workflow that combines Monte Carlo modeling (MC), gaussian combination, and additive noise to create synthetic dataset of volar fingertip PPG waveforms representative of a diverse cohort. First, MC is used to determine PPG amplitude across age, skin tone, and device wavelength. Then, gaussian combination generates accurate PPG waveforms, and signal processing enables data filtration and feature extraction. We improve the limitations of current synthetic PPG frameworks by enabling inclusion of physiological and anatomical effects from body site, skin tone, and age. We then show how the datasets can be used to examine effects of device characteristics such as wavelength, analog to digital converter specifications, filtering method, and feature extraction. Lastly, we demonstrate the use of this framework to show the insensitivity of a support vector machine predictive algorithm compared to a neural network and bagged trees algorithm.

Inorganic-Organic Interpenetrating Network Hydrogels as Tissue-Integrating Luminescent Implants: Physicochemical Characterization and Preclinical Evaluation.

Sensors capable of accurate, continuous monitoring of biochemistry are crucial to the realization of personalized medicine on a large scale. Great strides have been made to enhance tissue compatibility of long-term in vivo biosensors using biomaterials strategies such as tissue-integrating hydrogels. However, the low level of oxygen in tissue presents a challenge for implanted devices, especially when the biosensing function relies on oxygen as a measure-either as a primary analyte or as an indirect marker to transduce levels of other biomolecules. This work presents a method of fabricating inorganic-organic interpenetrating network (IPN) hydrogels to optimize the oxygen transport through injectable biosensors. Capitalizing on the synergy between the two networks, various physicochemical properties (e.g., swelling, glass transition temperature, and mechanical properties) are shown to be independently adjustable while maintaining a 250% increase in oxygen permeability relative to poly(2-hydroxyethyl methacrylate) controls. Finally, these gels, when functionalized with a Pd(II) benzoporphyrin phosphor, track tissue oxygen in real time for 76 days as subcutaneous implants in a porcine model while promoting tissue ingrowth and minimizing fibrosis around the implant. These findings support IPN networks for fine-tuned design of implantable biomaterials in personalized medicine and other biomedical applications.

Silicone-containing thermoresponsive membranes to form an optical glucose biosensor.

Glucose biosensors that could be subcutaneously injected and interrogated without a physically connected electrode and transmitter affixed to skin would represent a major advancement in reducing the user burden of continuous glucose monitors (CGMs). Towards this goal, an optical glucose biosensor was formed by strategically tailoring a thermoresponsive double network (DN) membrane to house a phosphorescence lifetime-based glucose sensing assay. This membrane was selected based on its potential to exhibit reduced biofouling via 'self-cleaning' due to cyclical deswelling/reswelling in vivo. The membrane was strategically tailored to incorporate oxygen-sensitive metalloporphyrin phosphor, Pd meso-tetra(sulfophenyl)-tetrabenzoporphyrin ([PdPh4(SO3Na)4TBP]3) (HULK) and glucose oxidase (GOx). Specifically, electrostatic interactions and colvalent bonds were used to stabilize HULK and GOx within the membrane, respectively. Enhancing the oxygen permeability of the membrane was necessary to achieve sensitivity of HULK/GOx to physiological glucose levels. Thus, silicone microparticles were incorporated at two concentrations. Key properties of SiHy-0.25 and SiHy-0.5 microparticle-containing compositions were compared to a control having no microparticles (SiHy-0). The discrete nature of the silicone microparticles maintained the desired thermosensitivity profile and did not impact water content. While the modulus decreased with silicone microparticle content, membranes were more mechanically robust versus a conventional hydrogel. SiHy-0.25, owing to apparent phase separation, displayed greater glucose diffusion and oxygen permeability versus SiHy-0.5. Furthermore, SiHy-0.25 biosensors exhibited the greatest glucose sensitivity range of 100 to 300 mg dL-1versus only 100 to 150 mg dL-1 for both SiHy-0 and SiHy-0.5 biosensors.