Progress in the Development of Intrinsically Conducting Polymer Composites as Biosensors

Biosensors are analytical devices which find extensive applications in fields such as the food industry, defense sector, environmental monitoring, and in clinical diagnosis. Similarly, intrinsically conducting polymers (ICPs) and their composites have lured immense interest in bio‐sensing due to their various attributes like compatibility with biological molecules, efficient electron transfer upon biochemical reactions, loading of bio‐reagent, and immobilization of biomolecules. Further, they are proficient in sensing diverse biological species and compounds like glucose (detection limit ≈0.18 nm ), DNA (≈10 pm ), cholesterol (≈1 µm ), aptamer (≈0.8 pm ), and also cancer cells (≈5 pm mL^?1) making them a potential candidate for biological sensing functions. ICPs and their composites have been extensively exploited by researchers in the field of biosensors owing to these peculiarities; however, no consolidated literature on the usage of conducting polymer composites for biosensing functions is available. This review extensively elucidates on ICP composites and doped conjugated polymers for biosensing functions of copious biological species. In addition, a brief overview is provided on various forms of biosensors, their sensing mechanisms, and various methods of immobilizing biological species along with the life cycle assessment of biosensors for various biosensing applications, and their cost analysis.     

Intrinsic disorder as a generalizable strategy for the rational design of highly responsive,allosterically cooperative receptors

Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. Such control could likewise be of utility in artificial biotechnologies, such as biosensors, genetic logic gates, and “smart” materials, in which highly responsive behavior is of value. In nature, the control of molecular responsiveness is often achieved using “Hill-type” cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. Here, we use an intrinsic-disorder–based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. To do so, we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. The first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a preformed site. Using this approach, we have rationally introduced cooperativity into three unrelated DNA aptamers, achieving in the best of these a Hill coefficient experimentally indistinguishable from the theoretically expected maximum. The extent of cooperativity and thus the steepness of the binding transition are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy.The ability to control the shape and midpoint of binding curves is critical to nature’s ability to optimize many cellular processes (1). One of the most widely used mechanisms by which nature so tunes the behavior of her receptors is allostery, in which the binding of one ligand alters the affinity with which subsequent ligands bind. Allostery comes in two “flavors.” Heterotropic allostery, in which the two ligands differ, provides a means of shifting the midpoint of a binding curve to higher or lower target concentrations without changing the curve’s intrinsically hyperbolic shape and thus without altering its sensitivity to small changes in the relative concentration of its molecular target (Fig. 1, Left). An example is the binding of bisphosphoglycerate to mammalian hemoglobin, which decreases the protein’s affinity for oxygen, thus pushing its binding curve to higher concentrations and enhancing oxygen transport efficiency, while leaving the intrinsic shape of its binding curve unaltered. Homotropic allostery, in contrast, occurs when the ligands are the same; that is, when the binding of one copy of a ligand changes the affinity with which subsequent copies of the same molecule bind. This mechanism, commonly referred to as “cooperativity,” changes not only the placement but also the shape of the binding curve, producing either a more responsive, higher-order dependence on ligand concentration (positive cooperativity) (Fig. 1, Right) or a less responsive, lower-order dependence (negative cooperativity). Like heterotropic allostery, cooperativity is also seen in the function of hemoglobin; the protein uses this mechanism to bind four oxygen molecules in a positively cooperative, approximately “all-or-nothing” fashion, steepening its binding curve and enhancing its ability to deliver oxygen over the rather modest concentration gradient present between the lungs and the peripheral tissues.Open in a separate windowFig. 1.Nature often controls the shape and position of ligand–response curves via allostery. (Left) In heterotropic allostery, the binding of one ligand to a receptor increases or decreases the affinity with which a second, different ligand binds, shifting the placement of the binding curve without altering its shape and thus without altering the width of its useful dynamic range (shaded boxes) or, in turn, its sensitivity to small changes in target concentration. (Right) In homotropic allostery, in contrast, the binding of one copy of target ligand changes the affinity with which additional copies of the same ligand bind, altering both the placement and the shape of the binding curve. The latter effect allows the system to respond more (positive cooperativity) or less (negative cooperativity) sensitively to changes in target ligand concentration. For positive cooperativity, receptor occupancy is a higher (than unity) order function of target concentration, with the exponent, n_H, being known as the Hill coefficient.The ubiquity with which nature exploits homotropic and heterotropic allostery has motivated efforts to rationally engineer these mechanisms into biomolecular receptors normally lacking them, both to test our understanding of the principles underlying these effects and to harness them to improve the utility of artificial biotechnologies. The rational introduction of heterotropic allostery into otherwise nonallosteric receptors, for example, has seen significant prior exploration (e.g., refs. 2–8), with both mechanical coupling (e.g., refs. 2 and 5–8) and mutually exclusive folding (e.g., refs. 3 and 4) approaches all having been used to successfully introduce this useful mechanism into a range of protein- and nucleic acid-based receptors. The design of allosterically cooperative receptors, in contrast, has seen far less success. That is, although a handful of examples of rationally designed cooperativity have been reported to date (9–12), no general approach has previously been reported by which such behavior can be rationally introduced into any arbitrarily complex biomolecule. This failure has limited the extent to which cooperativity, which could provide a powerful means of improving the ability of artificial biotechnologies to respond to small changes in molecular concentration (9, 13), can be applied in applications, such as biosensing (14, 15), “smart” drug delivery materials (16, 17), and molecular (18) and genetic (19) logic gates, in which such enhanced responsiveness would be of value.Two reasons account for why, despite its underlying simplicity and elegance, achieving the rational design of positive cooperativity has proven far from straightforward. First, to achieve the effect requires the creation of systems in which a higher affinity site is occupied only after a lower affinity site (which would normally be filled only at higher ligand concentrations) that binds the same ligand is already filled. This contrasts sharply with heterotropic allostery, in which the two binding sites typically exhibit little if any cross-reactivity. Second, all of the binding sites of a cooperative receptor recognize copies of the same ligand, rendering it more difficult to alter the affinity of one independently of that of the others. This is again in contrast to heterotropic allostery, in which each binding site is chemically distinct, allowing each to be independently optimized. Given these difficulties, and given the relative infancy of biomolecular design efforts (20–22), the ability to perform the structure-based design of cooperativity appears beyond current capabilities except for the simplest, most well-understood receptors (9–12). Here, however, we use an approach to the rational engineering of allosterically cooperative receptors that does not require detailed, structure-based design. Indeed, our approach is so simple that it can be performed, as demonstrated here, even in the absence of detailed knowledge of the parent receptor’s structure.Our design approach is inspired by intrinsically disordered proteins, proteins that are normally unfolded and only fold upon binding their target ligand. Specifically, both theoretical (23) and experimental (24, 25) studies have demonstrated that the global conformation change these proteins undergo upon an initial ligand binding event provides a convenient means of preorganizing a second, distal ligand binding site. This improves the affinity of the second binding event (because binding need no longer pay the unfavorable cost associated with folding), leading to positive allosteric behavior. Ferreon et al. (24), for example, have shown that the intrinsically disordered oncoprotein adenovirus early region 1A (E1A) folds upon binding either of its two (different) target ligands (CREB binding protein or retinoblastoma protein), thus increasing the affinity with which the second ligand binds and rendering the system heterotropically allosteric. In addition, Furukawa et al. (25) have shown that the partially intrinsically disordered protein STIM 1 exhibits strongly homotropic allosteric binding to calcium. Here, we use this same mechanism to rationally introduce cooperativity into a number of normally noncooperative aptamers (DNA-based receptors often adopting complex tertiary folds), thus producing steeper, more responsive binding curves than those seen for the unmodified parent molecule.     

中药衍生碳点研究进展

中药炭药及中药提取物经高温炭化会产生中药炭质药物纳米粒,其过程与碳量子点(carbon quantum dots,CDs)的制备工艺类似.由于粒径小于10nm,称之为中药衍生碳量子点.炭药的质量标准是存性,以中药为原材料制备而成的CDs,不仅具有止血、抗肿瘤等功效,而且具有低毒、水溶性好、生物相容性高、光致...     

Nucleic acid detection technologies and marker molecules in bacterial diagnostics

There is a growing need for quick and reliable methods for microorganism detection and identification worldwide. Although traditional culture-based technologies are trustworthy and accurate at a relatively low cost, they are also time- and labor-consuming and are limited to culturable bacteria. Those weaknesses have created a necessity for alternative technologies that are capable for faster and more precise bacterial identification from medical, food or environmental samples. The most common current approach is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to the specific nucleic acid profiles that are present. This review aims to give an up-to-date overview of different nucleic acid target sequences and respective analytical technologies.     

Advanced Optogenetic-Based Biosensing and Related Biomaterials

The ability to stimulate mammalian cells with light, brought along by optogenetic control, has significantly broadened our understanding of electrically excitable tissues. Backed by advanced (bio)materials, it has recently paved the way towards novel biosensing concepts supporting bio-analytics applications transversal to the main biomedical stream. The advancements concerning enabling biomaterials and related novel biosensing concepts involving optogenetics are reviewed with particular focus on the use of engineered cells for cell-based sensing platforms and the available toolbox (from mere actuators and reporters to novel multifunctional opto-chemogenetic tools) for optogenetic-enabled real-time cellular diagnostics and biosensor development. The key advantages of these modified cell-based biosensors concern both significantly faster (minutes instead of hours) and higher sensitivity detection of low concentrations of bioactive/toxic analytes (below the threshold concentrations in classical cellular sensors) as well as improved standardization as warranted by unified analytic platforms. These novel multimodal functional electro-optical label-free assays are reviewed among the key elements for optogenetic-based biosensing standardization. This focused review is a potential guide for materials researchers interested in biosensing based on light-responsive biomaterials and related analytic tools.     

Comprehensive pregnancy monitoring with a network of wireless,soft, and flexible sensors in high- and low-resource health settings

Vital signs monitoring is a fundamental component of ensuring the health and safety of women and newborns during pregnancy, labor, and childbirth. This monitoring is often the first step in early detection of pregnancy abnormalities, providing an opportunity for prompt, effective intervention to prevent maternal and neonatal morbidity and mortality. Contemporary pregnancy monitoring systems require numerous devices wired to large base units; at least five separate devices with distinct user interfaces are commonly used to detect uterine contractility, maternal blood oxygenation, temperature, heart rate, blood pressure, and fetal heart rate. Current monitoring technologies are expensive and complex with implementation challenges in low-resource settings where maternal morbidity and mortality is the greatest. We present an integrated monitoring platform leveraging advanced flexible electronics, wireless connectivity, and compatibility with a wide range of low-cost mobile devices. Three flexible, soft, and low-profile sensors offer comprehensive vital signs monitoring for both women and fetuses with time-synchronized operation, including advanced parameters such as continuous cuffless blood pressure, electrohysterography-derived uterine monitoring, and automated body position classification. Successful field trials of pregnant women between 25 and 41 wk of gestation in both high-resource settings (n = 91) and low-resource settings (n = 485) demonstrate the system’s performance, usability, and safety.

Pregnancy is experienced by more than 200 million people annually (1). Although pregnancy, labor, and delivery are common events, there remains a continued risk of catastrophic outcomes, with 23.8 maternal deaths per 100,000 births in the United States and 462 deaths per 100,000 births in low-income countries (2). Accordingly, ∼295,000 women die as a result of childbirth complications each year, with 94% of these deaths occurring in low- and middle-income countries (LMICs) (3). The most common causes of maternal morbidity and mortality—hemorrhage, hypertensive disorders, infection, and sepsis—are often preceded by predictable vital signs perturbations in both high-resource and low-resource settings, and early intervention is key to improving outcomes (4–9).Standard monitoring systems have changed minimally over the past 30 y (10). The most common systems for monitoring fetal heart rate (FHR) and uterine contractions during labor are rigid tocodynamometer systems (each typically 7- to 9-cm diameter in size) affixed to a patient’s abdomen with straps that have accompanying wires tethered to a wall-mounted base unit. A Doppler sensor measures FHR and a tocodynamometer measures uterine contraction. This limits a person’s mobility and comfort during labor. Unsurprisingly, pregnancy monitoring with cardiotocography (CTG) is therefore largely limited to intrapartum monitoring, facilitated by a hospital’s infrastructure. Additional monitoring equipment designed to assess maternal or fetal status, such as pulse oximetry, blood pressure (BP) monitors, and electrocardiography (ECG), are typically used in a noncontinuous manner and place a relatively high burden on providers (11). The failure to recognize and act on deteriorating vital signs is identified as a key contributor to preventable maternal deaths (12, 13). Cumulatively, the high base costs, expensive consumables, and operational burden of existing monitoring systems often preclude deployment in LMIC settings where maternal morbidity is the highest. In many parts of the world, a partograph, a simple graphical representation of maternal and fetal progress through labor, remains the only available method used to track labor despite a lack of evidence of clinical benefit for laboring women (14). In more developed countries, commercial innovation and growth have produced multiple home pregnancy monitoring systems. The Invu (formerly PregSense) from Nuvo Inc. and Bloomlife Pregnancy Tracker from Bloomlife Inc. aspire to bring the same care and attention available in the labor ward to the home. Such platforms, however, are not sufficiently comprehensive to replicate full clinical monitoring. The Invu reports both FHR and maternal HR but does not capture uterine contractions (15). The Bloomlife Pregnancy Tracker captures electrohysterography (EHG) without fetal ECG (16). Other systems, such as the Novii Wireless Patch System from Monica Healthcare, are available to acquire fetal ECG, but such systems alone cannot monitor nonsingleton pregnancies and are limited to term pregnancies (at least 37 wk of gestation) (17). While these devices excel in capitalizing on fetal measurements, they often overlook the importance of measuring core maternal vital signs of laboring women. Beyond these specific efforts, broader advances in both wearables, flexible electronics, and analytics have demonstrated the ability to monitor both traditional vital signs and novel digital biomarkers which hold future relevance for women’s health (18–23). Herein, we present a comprehensive system able to monitor all core vital signs for both the fetus and mother in the form of soft, flexible electronics with advanced analytics and wireless operation validated for performance in both low-and high-resource healthcare settings.There is a critical need for new classes of advanced maternal monitoring systems that offer comprehensive and continuous clinical-grade vital signs measurements, advanced monitoring capabilities to support clinical decision making, and operability in both high-resource and low-resource settings. We present an integrated monitoring platform leveraging advanced soft electronics, wireless connectivity, and compatibility with a wide range of low-cost mobile devices. This platform contains three flexible, soft, and low-profile sensors that offer comprehensive vital signs monitoring for both a woman and a fetus with time synchronized operation, allowing for advanced parameter assessment, such as continuous cuffless BP, EHG-derived uterine monitoring, and automated body position classification. The data streams generated by this system are integrated with the cloud, thereby allowing for future machine-learning opportunities to predict maternal or neonatal deterioration. Successful field trials among pregnant women between 25 and 41 wk of gestation (n = 576) in both high-resource (Chicago, IL) and low-resource (Lusaka, Zambia) settings for pregnant women demonstrate the system’s performance and usability.     

Development of Novel Glucose Sensing Fluids with Potential Application to Microelectromechanical Systems-Based Continuous Glucose Monitoring

Background

We have previously presented a microelectromechanical systems (MEMS) viscometric sensor for continuous glucose monitoring. The sensing fluid used therein was based on protein concanavalin A, which is known to have significant drawbacks, such as immunotoxicity and instability. To address this issue, a stable, biocompatible polymeric sensing fluid has been developed.

Methods

In the polymeric sensing system, glucose reversibly formed strong ester bonds with the phenylboronic acid moiety on the poly(acrylamide-ran-3-acrylamidophenylboronic acid) (PAA-ran-PAAPBA) polymer backbone, resulting in cross-linking of the copolymers and an increase in the solution viscosity. The copolymers were synthesized via classic free radical copolymerization processes. The viscosity of the PAA-ran-PAAPBA, dissolved in phosphate-buffered saline buffer and in the presence of glucose at physiologically relevant concentrations, was measured by an Ubbelodhe viscometer and a prototype MEMS viscometric device.

Results

Experimental results showed that the polymer molecular weight and composition depended on the solvent quantity, while the sensing fluid viscosity was determined by the polymer molecular weight and percentage composition of PAAPBA. The study of the temperature effect on viscosity showed that the polymer sensed glucose effectively under physiological conditions, although the high temperature lowered its sensitivity. Through proper adjustment of these parameters, a distinctive viscosity increase was observed when the glucose concentration increased from 0 to 450 mg/dl, which was detectable by our prototype MEMS device.

Conclusions

We have successfully developed a stable, biocompatible polymeric system for the sensitive detection of glucose. MEMS experiments demonstrated that the sensing fluid was able to sense glucose at different concentrations. This sensing system can potentially enable highly reliable, continuous monitoring of glucose in interstitial fluid from subcutaneous tissue.     

Predicting Glucose Sensor Behavior in Blood Using Transport Modeling: Relative Impacts of Protein Biofouling and Cellular Metabolic Effects

Background

Tissue response to indwelling glucose sensors remains a confounding barrier to clinical application. While the effects of fully formed capsular tissue on sensor response have been studied, little has been done to understand how tissue interactions occurring before capsule formation hinder sensor performance. Upon insertion in subcutaneous tissue, the sensor is initially exposed to blood, blood borne constituents, and interstitial fluid. Using human whole blood as a simple ex vivo experimental system, the effects of protein accumulation at the sensor surface (biofouling effects) and cellular consumption of glucose in both the biofouling layer and in the bulk (metabolic effects) on sensor response were assessed.

Methods

Medtronic MiniMed SofSensor glucose sensors were incubated in whole blood, plasma-diluted whole blood, and cell-free platelet-poor plasma (PPP) to analyze the impact of different blood constituents on sensor function. Experimental conditions were then simulated using MATLAB to predict the relative impacts of biofouling and metabolic effects on the observed sensor responses.

Results

Protein biofouling in PPP in both the experiments and the simulations was found to have no interfering effect upon sensor response. Experimental results obtained with whole and dilute blood showed that the sensor response was markedly affected by blood borne glucose-consuming cells accumulated in the biofouling layer and in the surrounding bulk.

Conclusions

The physical barrier to glucose transport presented by protein biofouling does not hinder glucose movement to the sensor surface, and the consumption of glucose by inflammatory cells, and not erythrocytes, proximal to the sensor surface has a substantial effect on sensor response and may be the main culprit for anomalous sensor behavior immediately following implantation.     

Water toxicity detection by a panel of stress-responsive luminescent bacteria

A panel of Escherichia coli strains harbouring different stress-responsive promoters fused to a lux reporter system was used to assess the potential toxicity of 17 unknown model water samples. Using liquid cultures, nine out of 14 toxic samples were properly identified as toxic, whereas five were false negatives. All three non-toxic controls were identified correctly (no false positives). Two strains containing promoter-lux fusions were also tested when immobilized onto fibre-optic tips. One genotoxic sample and six toxic samples were correctly identified in this manner. The potential advantages and limitations in the use of genetically engineered bacteria as biosensors for water toxicity are discussed in view of these results.