Artificial Interstitial Fluid Research Articles

P-696 Continuous monitoring of female hormones as a tool to improve IVF treatment’s efficacy

Abstract Study question Is it possible to employ an unobtrusive and injectable biosensor for real-time remote monitoring of patient’s hormone levels (LH, P4, E2) during an ART cycle? Summary answer In-situ implantable biosensors can effectively monitor clinically relevant hormone levels in real-time to improve ART cycle. What is known already Worldwide 48.5 million couples suffer from infertility. Hormonal imbalances can impede pregnancy by compromising the intricate processes of the reproductive system, affecting ovulation, egg quality, fallopian tube function, and endometrial receptivity. In the UK, assisted reproductive treatments (ART) have surged ten-fold in the last 20 years. ART success tremendously depends on the accurate measurement of hormone levels, namely Luteinizing Hormone (LH), oestradiol (E2), and progesterone (P4). These hormone levels change and pulsate during each patient cycle, requiring women to undergo a blood draw and endocrinology analysis every 2-3 days, making ART treatment a highly invasive and time consuming method. Study design, size, duration In order to determine the feasibility of the study question, Impli designed a minimally invasive implantable biosensor to continuously monitor essential hormones for women’s fertility such as LH, P4 and E2 in order to enhance the quality of treatment and improve the ART experience. An implantable electrochemical aptamer-based biosensors has been developed using cutting-edge electronics and electrochemical technologies. This minimally invasive biosensor allows patients to monitor their hormone levels in real-time during ART without venipuncture. Participants/materials, setting, methods The implantable biosensor contains the biorecognition interface, designed using the DNA-based aptamers coupled to the detection gold working electrodes. When the biosensor is in touch with the sample, hormones bind to the respective aptamers, which leads to changes in the electrical properties of the gold electrode. The changes are then monitored and calibrated through sensitive electrochemical techniques such as differential pulse voltammetry (DPV), using CHI potentiostat. Main results and the role of chance The biosensor was tested in vitro in artificial interstitial fluid (ISF) containing the range of 1 pg/ml to 1 µg/ml of hormones yielding a limit of detection (LOD) of 0.46 pg/ml, 1.15 pg/ml, and 0.58 pg/ml for E2, P4 and LH, respectively. Our data was validated using ELISA assays. A notably high linear correlation coefficient (R2) of approximately 0.9 was observed between the signals obtained from the ELISA and the biosensors, confirming the reliability of the aptamer biosensors for hormone analysis. The stability of the biosensor was also investigated over one week. The DPV technique was employed to assess the cross-reactivity of potential interferences with each biosensor. The results revealed that each biosensor exhibited notable selectivity towards its respective analyte over other interferences, at significantly higher concentrations. The biosensor was also characterised by microscopy techniques such as SEM and Raman analysis. Limitations, reasons for caution This novel implantable biosensor technology requires further upcoming validation in animal models to assess its ability for widespread use. Once validated, the appropriate human clinical trials will be undertaken. Wider implications of the findings The development and implementation of an unobtrusive implantable remote and continuous monitoring system, capturing hormone concentrations that are vital for success in an ART procedure, could transform the industry and positively influence patient’s access to care and the patient journey. Trial registration number No clinical trial

The 2.5th generation enzymatic sensors based on the construction of quasi-direct electron transfer type NAD(P)-Dependent dehydrogenases

We introduce a versatile method to convert NAD+ or NADP+ -dependent dehydrogenases into quasi-direct electron transfer (quasi-DET)-type dehydrogenases, by modifying with a mediator on the enzyme surface toward the development of 2.5th generation enzymatic sensors. In this study, we use β-hydroxybutyrate (BHB) dehydrogenase (BHBDh) from Alcaligenes faecalis (AfBHBDh) as a representative NAD+ or NADP+ -dependent dehydrogenase. BHBDhs are important in ketone monitoring, especially for the diagnosis of diabetic ketoacidosis. We modified AfBHBDh with a thiol-reactive phenazine ethosulfate (trPES). We designed, constructed, and modified mutant BHBDhs harboring cysteine residues within 20 Å from the C4 nicotinamide in NAD+/NADH. Mutants Ser65Cys, Thr96Cys, and Lys106Cys showed indistinguishable catalytic activities from the wild-type enzyme, even after trPES modification. These trPES-modified mutants were immobilized on gold disk electrodes via amine coupling with succinimide-groups of dithiobis (succinimidyl hexanoate) self-assembled monolayers for electrochemical measurements. Considering there is a wide range of BHB concentrations, we exploited the linear regression in log scales. The linear range for the sensors with trPES-modified BHBDh mutants Ser65Cys, Thr96Cys, and Lys106Cys were 0.1–4.0 mM in both buffer solution and artificial interstitial fluid (ISF). They have limits of detection of 0.047 mM for Ser65Cys, 0.15 mM for Thr96Cys, and 0.060 mM for Lys106Cys in buffer solution, and 0.12 mM, 0.089 mM, and 0.044 mM in artificial ISF, respectively. These results indicate that redox mediator modification of NAD(P)-dependent dehydrogenases converts them into quasi-DET-type dehydrogenases, thereby enabling their utilization in 2.5th generation enzymatic sensors, which will facilitate the construction of enzymatic sensors suitable for continuous monitoring systems.

Biopolymer-protected graphene-Fe3O4 nanocomposite based wearable microneedle sensor: toward real-time continuous monitoring of dopamine.

Neurological disorders can occur in the human body as a result of nano-level variations in the neurotransmitter levels. Patients affected by neuropsychiatric disorders, that are chronic require continuous monitoring of these neurotransmitter levels for effective disease management. The current work focus on developing a highly sensitive and personalized sensor for continuous monitoring of dopamine. Here we propose a wearable microneedle-based electrochemical sensor, to continuously monitor dopamine in interstitial fluid (ISF). A chitosan-protected hybrid nanomaterial Fe3O4-GO composite has been used as a chemical recognition element protected by Nafion antifouling coating layer. The morphological and physiochemical characterizations of the nanocomposite were carried out with XRD, XPS, FESEM, EDAX and FT-IR. The principle of the developed sensor relies on orthogonal detection of dopamine with square wave voltammetry and chronoamperometric techniques. The microneedle sensor array exhibited an attractive analytical performance toward detecting dopamine in phosphate buffer and artificial ISF. The limit of detection (LOD) of the developed sensor was observed to be low, 90 nM in square wave voltammetry and 0.6 μM in chronoamperometric analysis. The practical applicability of the microneedle sensor array has been demonstrated on a skin-mimicking phantom gel model. The microneedle sensor also exhibited good long-term storage stability, reproducibility, and sensitivity. All of these promising results suggest that the proposed microneedle sensor array could be reliable for the continuous monitoring of dopamine.

Reduced graphene oxide-functionalized polymer microneedle for continuous and wide-range monitoring of lactate in interstitial fluid

Despite substantial developments in minimally invasive lactate monitoring microneedle electrodes, most such electrode developments have focused on either sensitivity or invasiveness while ignoring a wide range of detection, which is the most important factor in measuring the normal range of lactate in interstitial fluid (ISF). Herein, we present a polymer-based planar microneedle electrode fabrication using microelectromechanical and femtosecond laser technology for the continuous monitoring of lactate in ISF. The microneedle is functionalized with two-dimensional reduced graphene oxide (rGO) and electrochemically synthesized platinum nanoparticles (PtNPs). A particular quantity of Nafion (1.25 wt%) is applied on top of the lactate enzyme to create a diffusion-controlled membrane. Due to the combined effects of the planar structure of the microneedle, rGO, and membrane, the biosensor exhibited excellent linearity up to 10 mM lactate with a limit of detection of 2.04 μM, high sensitivity of 43.96 μA mM−1cm−2, a reaction time of 8 s and outstanding stability, selectivity, and repeatability. The feasibility of the microneedle is evaluated by using it to measure lactate concentrations in artificial ISF and human serum. The results demonstrate that the microneedle described here has great potential for use in real-time lactate monitoring for use in sports medicine and treatment.

Development of a Self-Powered Microneedle Based Wearable Electrochemical Sensor for Glucose Monitoring

Diabetes is increasingly being recognized as a worldwide public health concern owing to the affected rate of the patients in the past years. One of the major barriers to the disease control is the lack of sensitive and reliable point-of-care device that can provide quantitative diagnosis and monitoring. The wearable sensing technology has the potential of revolutionizing the precision medicine and personalized healthcare system. Within the emerging class of wearables, the microneedle-based sensors play a key role in combining the advantages of monitoring the analytes in interstitial fluid (ISF) and minimally invasive skin pricking. However, such devices require appropriate power sources to realize their full capacity, and the increasing power costs, global warming, and environmental pollution are certain aspects that needs to be pondered upon. Researches are dedicated on power generators with focus on wearable energy harvesters to address the issue of power supply. Thermoelectric generators (TEGs) have the ability to generate power by transforming thermal energy to electrical power output. Herein, we have developed a microneedle based enzymatic, electrochemical glucose sensing system powered by TEG utilizing the body heat. 1 mm stainless steel microneedles were sputtered with gold and platinum, and further coated with graphene paste, glucose oxidase, poly-hydroxyethyl methacrylate, and nafion. The fabricated device exhibited a good sensitivity, and a wide linear range. According to the World Health Organization (WHO), the normal glucose level in human body are between 4.0 mM to 5.6 mM, and our device has shown a concentration dependent output current signal from 4.0 mM to 24.0 mM. The device has also exhibited excellent detection results in real-sample (artificial ISF) in vitro study as well as in vivo analysis performed in 12 – 15 weeks SD rats. The device is completely self-powered where the TEG derives power from the temperature gradient between the body heat and the exposed environment. The novelty of this work lies in integrating the microneedle-based sensing device with the TEG as the power source. The results indicate a promising self-powered sensor with the renewable and sustainable source of power ensuring continuous operation.

Microneedle array-based electrochemical sensor functionalized with SWCNTs for the highly sensitive monitoring of MDMA in interstitial fluid

Illicit drug consumption constitutes a great concern worldwide due to its increased spread and abuse, and the negative consequences exerted on society. For instance, 3,4-methylenedioxymethamphetamine (MDMA), a synthetic amphetamine-type substance, was abused by 20 million people worldwide in 2020. This psychoactive substance exerts a myriad of effects on the human body being dangerous for the consumer’s health. Besides, MDMA has been used in the treatment of some psychiatric conditions. Therefore, the development of wearable devices for MDMA sensing in biological fluids is of great importance for forensic toxicology (e.g., monitoring of patients with suspected or known MDMA consumption) as well as for therapeutic management of patients. Herein, we report the development of a wearable electrochemical platform based on a hollow microneedle (MN) array sensor for the monitoring of MDMA in the interstitial fluid by square-wave voltammetry. First, the holes of the MN array were modified with conductive pastes to devise a MN patch with a three-electrode system. Subsequently, the functionalization of the working electrode with nanomaterials enhanced MDMA detection. Thereafter, analytical parameters were evaluated exhibiting a slope of 0.05 µA µM−1 within a linear range from 1 to 50 µM and a limit of detection of 0.75 µM in artificial interstitial fluid. Importantly, critical parameters such as selectivity, piercing capability, temperature, reversibility and stability were assessed. Overall, the obtained MN sensor exhibited excellent analytical performance, making it a promising tool for MDMA tracking in interstitial fluid for individuals on probation or under therapeutic treatment.

A Highly Sensitive and Specific Photonic Crystal-Based Opioid Sensor with Rapid Regeneration.

Opioid misuse and overdose have caused devastating public health challenges and economic burdens, calling for the need of rapid, accurate sensitive opioid sensors. Here, we report a photonic crystal-based opioid sensor in the total internal reflection configuration, providing label-free, rapid, quantitative measurements through change of the refractive index. The one-dimensional photonic crystal with a defect layer that is immobilized with opioid antibodies acts as a resonator with an open microcavity. The highly accessible structure responds to analytes within a minute after the aqueous opioid solution is introduced, achieving the highest sensitivity of 5688.8 nm/refractive index unit (RIU) at the incident angle of 63.03°. Our sensor shows a limit of detection (LOD) of 7 ng/mL for morphine in phosphate-buffered saline (PBS, pH 7.4) solutions, well below the required clinical detection limit, and an LOD of 6 ng/mL for fentanyl in PBS, close to the clinical requirement. The sensor can selectively detect fentanyl from a mixture of morphine and fentanyl and be regenerated in 2 min with up to 93.66% recovery rate after five cycles. The efficacy of our sensor is further validated in artificial interstitial fluid and human urine samples.

Electrochemical detection of cholesterol in human biofluid using microneedle sensor.

The development of a straightforward, economical, portable, and highly sensitive sensing platform for the rapid detection of cholesterol is desirable for the early diagnosis of several pathologic conditions. In this work, we present a fascinating skin-worn microneedle sensor for monitoring cholesterol in interstitial fluid samples. The microneedle sensor was developed by incorporating platinum (Pt) and silver (Ag) wires within pyramidal microneedles containing a microcavity opening; cholesterol oxidase (ChOx) was coupled on the Pt transducer surface using bovine serum albumin and Nafion. Under optimal conditions, the enzymatic microneedle sensor exhibited high sensitivity (0.201 μA μM-1) towards cholesterol in buffer solution, with good linearity over the 1-20 μM range and a correlation coefficient of 0.9910. The analytical performance of the microneedle sensor was also investigated in artificial interstitial fluid and a skin-mimicking phantom gel; the sensor showed great potential for skin-worn/wearable applications with excellent linearity and a low detection limit. In addition, the developed microneedle sensor showed satisfactory stability and good selectivity towards cholesterol in the presence of potential interfering biomolecules, including glucose, lactic acid, uric acid, and ascorbic acid. This sensor exhibits enormous promise for straightforward, sensitive, and minimally invasive monitoring of cholesterol.

A paper-based colorimetric method for monitoring of lithium therapeutic levels.

Lithium remains the "gold standard" for both acute and maintenance treatment of bipolar disorder (BD), a serious life-long condition characterised by recurrent episodes of depressed and manic mood states. However, lithium has a very narrow therapeutic range (0.4-1.2 mmol L-1) and despite its effectiveness in preventing and reducing mood swings and suicidality, it is a potentially hazardous drug. While it is crucial to carefully monitor lithium plasma levels, the current techniques of lithium monitoring are cumbersome and require frequent blood tests with the consequent discomfort which results in patients evading treatment. Therefore, development of low-cost and facile lithium detection techniques that can be translated into point-of-care devices for personal monitoring will be a major advance in the management of BD. In the current study, we present colorimetric determination of lithium therapeutic levels utilizing test paper strips, based on its reaction with the chromogenic agent Quinizarin. Exposure of Quinizarin-dipped test papers to samples of interstitial fluid (ISF) or dH2O spiked with therapeutic concentrations of lithium resulted in colour changes that were monitored using optical spectroscopy. The acquired spectra from the test papers show spectral variations which are related to lithium concentrations in spiked samples of dh2O and artificial ISF with a coefficient of determination (R2) of 0.9 and 0.8, respectively. Altogether, the spectrophotometric and colorimetric analyses demonstrated strong correlations between the observed colour changes and the concentrations of lithium present in the sample. Therefore, this study has demonstrated that Quinizarin-treated cellulose-based papers are suitable for the precise detection of changes in lithium therapeutic levels. This method is simple and very convenient and serves as a foundation for the future development of a paper-based colorimetric sensor for monitoring of lithium therapeutic levels in ISF and other non-invasive biological fluids.

Microneedle-based nanoporous gold electrochemical sensor for real-time catecholamine detection

Dopamine (DA), epinephrine (EP), and norepinephrine (NEP) are the main catecholamine of clinical interest, as they play crucial roles in the regulation of nervous and cardiovascular systems and are involved in some brain behaviors, such as stress, panic, anxiety, and depression. Therefore, there is an urgent need for a reliable sensing device able to provide their continuous monitoring in a minimally invasive manner. In this work, the first highly nanoporous gold (h-nPG) microneedle-based sensor is presented for continuous monitoring of catecholamine in interstitial fluid (ISF). The h-nPG microneedle-based gold electrode was prepared by a simple electrochemical self-templating method that involves two steps, gold electrodeposition and hydrogen bubbling at the electrode surface, realized by sweeping the potential between + 0.8 V and 0 V vs Ag/AgCl for 25 scans in a 10 mM HAuCl4 solution containing 2.5 M NH4Cl, and successively applying a fixed potential of − 2 V vs Ag/AgCl for 60 s. The resulting microneedle-based h-nPG sensor displays an interference-free total catecholamine detection expressed as NEP concentration, with a very low LOD of 100 nM, excellent sensitivity and stability, and fast response time (< 4 s). The performance of the h-nPG microneedle array sensor was successively assessed in artificial ISF and in a hydrogel skin model at typical physiological concentrations.Graphical abstract

All-solid-state potassium-selective sensor based on carbon black modified thermoplastic electrode

Thermoplastic carbon composite electrodes have been introduced by our group as novel electrodes with improved electrochemical properties and previously applied for accurate detection of biomarkers using voltammetric methods. Because of their exceptional properties, we hypothesized they would work well as potentiometric ion-selective electrodes as well. In this work, potassium selective thermoplastic electrodes (TPEs) were developed for potentiometric detection for the first time and applied in biofluids. TPEs were fabricated using mixture of polystyrene and polycaprolactone and two types of graphite (MG1599 and Nano19) for comparison, and modified with carbon black via drop-casting. Carbon black modification was optimized in the presence of various numbers of carbon black layers, while ion selective membrane components were optimized at different component ratios. Under optimized conditions, TPEs exhibited Nernstian response to K+ with sensitivity of 59.3 ± 1.01 mV decade−1 and near-Nernstian response with sensitivity of 56.8 ± 1.7 mV decade−1 within a linear range of 1.0 × 10−4 M-1.0 × 10−1 M with a LOD of 1.0 × 10−4- M for Nano19- and MG1599-based TPEs, respectively. The K+-selective TPEs demonstrated rapid response (4 s) and excellent stability, repeatability and reproducibility. The applicability of electrodes were shown in human pooled urine and artificial interstitial fluid samples with potassium ion concentrations ranging from healthy and unhealthy levels. Therefore, the electrodes could be used in healthcare applications for accurate detection of K+ exists in complex matrix and can be easily adapted to simultaneously monitor other ions for diagnosis of diseases due to miniaturization of TPEs.

Wearable electrochemical microneedle sensing platform for real-time continuous interstitial fluid monitoring of apomorphine: Toward Parkinson management

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, impacting millions of people across the globe. For its management, pro-neurotransmitters or agonists are commonly being prescribed, which help to mitigate the symptoms. However, the progressiveness of PD and narrowing down of the drug therapeutic window eventually led to its ineffective management. Therefore, the requirement of continuous monitoring of administered drugs is of utmost need. In view of this, here, we demonstrate a microneedle-based wearable electrochemical sensor platform for the continuous monitoring of apomorphine (APO), a widely used therapeutic drug for Parkinson’s management. The APO sensor relies on a metallized carbon-paste packed hollow microneedle, operated using the square wave voltammetry (SWV) and chronoamperometry (CA) based transduction modes. Under the optimal conditions, the microneedle sensor offers sensitive detection of micromolar APO concentrations in artificial interstitial fluid (ISF) through the monitoring of the two oxidation peaks of the APO catechol and tertiary amine moieties (at 0.1 V and 0.7 V, respectively). The microneedle sensor exhibited a detection limit of 0.6 and 0.75 μM with the transduction techniques SWV and CA, respectively, and good operational stability as well as anti-biofouling protection in artificial-ISF medium. The specificity has been tested for the developed sensor in the presence of potential interfering molecules, where the developed sensor showed the selectivity towards APO. The attractive analytical performance demonstrated in the skin mimic environment makes the new wearable APO microneedle sensor a powerful candidate for continuous APO detection in PD management.

Optical Determination of Lithium Levels in Artificial Interstitial Fluid for Treatment Management of Bipolar Disorder.

Bipolar Disorder (BD), characterized by mood fluctuating between episodes of mood elevation and depression, is a leading cause of disability worldwide. Lithium continues to be prescribed as a first-line mood stabilizer for the management of BD. However, lithium has a very narrow therapeutic index and it is crucial to carefully monitor lithium plasma levels as concentrations greater than 1.2 mmol/L are potentially toxic and can be fatal. The current techniques of lithium monitoring are cumbersome and require frequent blood tests with the consequent discomfort which results in patients evading treatment. Dermal interstitial fluid (ISF), an underutilized information-rich biofluid, can be a proxy for direct blood sampling and allow lithium drug monitoring as its lithium concentration is proportional to the concentrations in blood. Therefore, in this study we seek to investigate the measurement of lithium therapeutic concentrations in artificial ISF. Our study employs a colorimetric method, based on the reaction between chromogenic agent Quinizarin and Li+ ion which can be detected using optical spectroscopy in the visible region (400-800 nm), to determine lithium levels in artificial ISF. The resulting spectra of our experiments show spectral variations which are related to lithium concentrations in spiked samples of artificial ISF, with a correlation coefficient (R) of 0.9. Future work will focus on investigating the feasibility of utilizing ISF for real-time and minimally-invasive lithium drug monitoring.

Electrochemical immunosensor for breast cancer biomarker detection using high-density silicon microneedle array

Electrochemical devices for transdermal monitoring of key biomarkers are the potential next frontier of wearable technologies for point-of-care disease diagnosis, including Cancer in which Cancer is the leading cause of death worldwide with estimated 10 million deaths in 2018 according to the World Health Organization and breast cancer is one of the five most common causes of cancer death with over two million cases recorded in 2018. Early diagnosis and prognosis based on monitoring of breast cancer biomarkers is of high importance. In this work, high-density gold coated silicon microneedle arrays (Au–Si-MNA) were simultaneously used as biomarker extraction platform and electrochemical transducer, enabling the selective immunocapture of epidermal growth factor receptor 2 (ErbB2), a key breast cancer biomarker, and its subsequent quantification. The analytical performance of the device was tested in artificial interstitial fluid exhibiting a linear response over a wide concentration range from 10 to 250 ng/mL, with a detection limit of 4.8 ng/mL below the biomarker levels expected in breast cancer patients. As a proof of concept, the immunosensor demonstrated its ability to successfully extract ErbB2 from a phantom gel mimicking the epidermis and dermis layers, and subsequently quantify it showing a linear range from 50 to 250 ng/mL and a detection limit of 25 ng/mL. The uniqueness of this sensing platform combining direct transdermal biomarker extraction and quantification opens up new avenues towards the development of high performing wearable point-of-care devices.

Electrochemical stability ofPEDOTfor wearableon‐skinapplication

AbstractConducting polymers are promising candidates for wearable devices due to mechanical flexibility combined with electroactivity. While electrochemical measurements have been adopted as a central transduction method in many on‐skin sensors, less studied is the stability of the active materials (in particular poly3,4‐ethylenedioxythiophene, PEDOT) in such systems, particularly for “on‐skin” applications. In this study, several different variants of doped PEDOT are fabricated and characterized in terms of their (electrical, physical, and chemical) stability in biological fluid. PEDOT doped with tosylate (TOS) or polystyrenesulfonate (PSS) are selected as prototypical forms of conducting polymers. These are compared with a new variant of PEDOT co‐doped with both TOS and PSS. Artificial interstitial fluid (aISF) loaded with 1% wt/vol bovine serum albumin is adopted as the testing medium to demonstrate the stability in dermal applications (i.e., conducting polymer microneedles or coatings on microneedles). A range of techniques such as cyclic voltammetry and electrochemical impedance spectroscopy are used to qualify and quantify the stability of the doped conducting polymers. Furthermore, this study is extended by using human skin lysate in the aISF to demonstrate proof‐of‐concept for stable use of PEDOT in wearable “on‐skin” electronics.

Transdermal Electrochemical Monitoring of Glucose via High‐Density Silicon Microneedle Array Patch

AbstractCurrent technology for blood glucose level monitoring is mainly based on the invasive finger‐prick extraction of a small drop of blood using a lancet and measured via a handheld glucometer, which is not conducive to continuous measurements. Interstitial fluid (ISF) is gaining attention as an alternative biofluid. Its biochemical composition is very similar to that of blood and it can be monitored in a continuous manner via minimally invasive methods that cause no pain and minimize any risk of infection. Herein, a microneedle array (MNA) based transdermal sensing system for the pain free monitoring of ISF glucose is presented. High‐density silicon microneedles (≈9500 microneedles cm−2) are used to prepare a three‐electrode patch for the electrochemical monitoring of glucose. The MNA glucose patch shows very good selectivity when tested in artificial ISF, with a sensitivity of 0.1622 µA mm−1 cm−2 and a detection limit of 0.66 mm. In vivo application of the microneedle array in mice shows that the ISF glucose concentrations obtained with the MNA sensor gave very good correlation with the blood glucose levels determined with a commercial glucometer. This microneedle‐based sensing system hence provides an alternative transdermal diagnostic tool to the invasive existing techniques.

Toward In Vivo Transdermal pH Sensing with a Validated Microneedle Membrane Electrode.

We present herein the most complete characterization of microneedle (MN) potentiometric sensors for pH transdermal measurements for the time being. Initial in vitro assessment demonstrated suitable analytical performances (e.g., Nernstian slope, linear range of response from 8.5 to 5.0, and fast response time) in both buffer media and artificial interstitial fluid (ISF). Excellent repeatability and reproducibility together with adequate selectivity and resiliency facilitate the appropriateness of the new pH MN sensor for transdermal ISF analysis in healthcare. The ability to resist skin insertions was evaluated in several ex vivo setups using three different animal skins (i.e., chicken, pork, and rat). The developed pH MN sensor was able to withstand from 5 to 10 repetitive insertions in all the skins considered with a minimal change in the calibration graph (<3% variation in both slope and intercept after the insertions). Ex vivo pH measurements were validated by determining the pH with the MN sensor and a commercial pH electrode in chicken skin portions previously conditioned at several pH values, obtaining excellent results with an accuracy of <1% and a precision of <2% in all cases. Finally, pH MN sensors were applied for the very first time to transdermal measurements in rats together with two innovative validation procedures: (i) measuring subcutaneous pH directly with a commercial pH microelectrode and (ii) collecting ISF using hollow MNs and then the pH measurement of the sample with the pH microelectrode. The pH values obtained with pH MN sensors were statistically more similar to subcutaneous measurements, as inferred by a paired sample t-test at 95% of confidence level. Conveniently, the validation approaches could be translated to other analytes that are transdermally measured with MN sensors.

Minimally invasive and continuous glucose monitoring sensor based on non-enzymatic porous platinum black-coated gold microneedles

Individuals with diabetes can considerably benefit from continuous glucose monitoring (CGM). Addressing this challenge, a proof-of-concept for a pain-free CGM was presented based on a highly porous platinum black (Pt-black)-modified non-enzymatic microneedle electrode arrays (MNEAs). Porous Pt-black was electrodeposited on microneedle substrates using potentiometry by employing a charge density of -2.5 mA cm˗2. The surface of the porous Pt-black-modified microneedles (MNs) was optimally packed using Nafion (Nf) biocompatible ionomer. The dimensions of the fabricated microneedles were 650 µm × 110 µm × 150 µm in height, width, and thickness, respectively. The surface morphology of the modified MNs was studied using scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX). The microneedle sensor showed a wide glucose dynamic range (1 − 20 mM) in phosphate buffer saline (PBS) and artificial interstitial fluid (ISF) in-vitro with good sensitivities (PBS: 5.786 µA mM−1 cm−2, ISF: 4.380 µA mM−1 cm−2), detection limits (PBS: 10 µM, ISF: 22.5 µM), stability (2% lost at the end of 7 h), selectivity, reproducibility (2.32% RSD), and a response time of 2 s with good glucose recoveries in ISF (98%-102.5%). The device exhibited good stability (7 days) as a sensor in-vivo and lost its functional activity after 7 days due to electrode biofouling.

Porous Platinum Black-Coated Minimally Invasive Microneedles for Non-Enzymatic Continuous Glucose Monitoring in Interstitial Fluid.

Individuals with diabetes can benefit considerably from continuous blood glucose monitoring. To address this challenge, a proof-of-concept was performed for continuous glucose monitoring (CGM) based on an enzymeless porous nanomaterial (pNM)-modified microneedle electrode array (MNEA). The pNM sensing layer was electrochemically deposited on MNs by applying a fixed negative current of −2.5 mA cm˗2 for 400 s. The pNM-modified MNEA was packed using a biocompatible Nafion ionomer. The fabricated MNEAs were 600 × 100 × 150 µm in height, width, and thickness, respectively. The surfaces of the modified MNs were characterized by scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). The fabricated MNEAs showed a wide dynamic range (1–30 mM) in phosphate-buffered saline (PBS) and in artificial interstitial fluid (ISF), with good sensitivities (PBS: 1.792 ± 0.25 µA mM−1 cm−2, ISF: 0.957 ± 0.14 µA mM−1 cm−2) and low detection limits (PBS: 7.2 µM, ISF: 22 µM). The sensor also showed high stability (loss of 3.5% at the end of 16 days), selectivity, and reproducibility (Relative standard deviations (RSD) of 1.64% and 0.70% for intra- and inter-assay, respectively) and a good response time (2 s) with great glucose recovery rates in ISF (98.7–102%).

Strategic design and improvement of the internal electron transfer of heme b domain-fused glucose dehydrogenase for use in direct electron transfer-type glucose sensors

A fusion enzyme composed of an Aspergillus flavus-derived flavin adenine dinucleotide glucose dehydrogenase (AfGDH) and an electron transfer domain of Phanerochaete chrysosporium-derived cellobiose dehydrogenase (Pcyb) was previously reported to show the direct electron transfer (DET) ability to an electrode. However, its slow intramolecular electron transfer (IET) rate from the FAD to the heme, limited the sensor signals. In this study, fusion FADGDH (Pcyb-AfGDH) enzymes were strategically redesigned by performing docking simulation, following surface-electrostatic potential estimation in the predicted area. Based on these predictions, we selected the amino acid substitution on Glu324, or on Asn408 to Lys to increase the positive charge at the rim of the interdomain region. Pcyb-AfGDH mutants were recombinantly produced using Pichia pastoris as the host microorganism, and their IET was evaluated. Spectroscopic observations showed that the Glu324Lys (E324K) and Asn408Lys (N408K) Pcyb-AfGDH mutants showed approximately 1.70- and 9.0-fold faster IET than that of wildtype Pcyb-AfGDH, respectively. Electrochemical evaluation revealed that the mutant Pcyb-AfGDH-immobilized electrodes showed higher DET current values than that of the wildtype Pcyb-AfGDH-immobilized electrodes at pH 6.5, which was approximately 9-fold higher in the E324K mutant and 15-fold higher in the N408K mutant, than in the wildtype. Glucose enzyme sensors employing N408K mutant was able to measure glucose concentration under physiological condition using artificial interstitial fluid at pH 7.4, whereas the one with wildtype Pcyb-AfGDH was not. These results indicated that the sensor employed the redesigned mutant Pcyb-AfGDH can be used for future continuous glucose monitoring system based on direct electron transfer principle. (247 words).