Multi-engineered Graphene Extended-Gate Field-Effect Transistor for Peroxynitrite Sensing in Alzheimer's Disease.

The expression of ß-amyloid peptides (Aß), a pathological indicator of Alzheimer's disease (AD), was reported to be inapparent in the early stage of AD. While peroxynitrite (ONOO-) is produced excessively and emerges earlier than Aß plaques in the progression of AD, it is thus significant to sensitively detect ONOO- for early diagnosis of AD and its pathological research. Herein, we unveiled an integrated sensor for monitoring ONOO-, which consisted of a commercially available field-effect transistor (FET) and a high-performance multi-engineered graphene extended-gate (EG) electrode. In the configuration of the presented EG electrode, laser-induced graphene (LIG) intercalated with MnO2 nanoparticles (MnO2/LIG) can improve the electrical properties of LIG and the sensitivity of the sensor, and graphene oxide (GO)-MnO2/Hemin nanozyme with ONOO- isomerase activity can selectively trigger the isomerization of ONOO- to NO3-. With this synergistic effect, our EG-FET sensor can respond to the ONOO- with high sensitivity and selectivity. Moreover, taking advantage of our EG-FET sensor, we modularly assembled a portable sensing platform for wireless tracking ONOO- levels in the brain tissue of AD transgenic mice at earlier stages before massive Aß plaques appeared, and we systematically explored the complex role of ONOO- in the occurrence and development of AD.

Tailoring Food Biopolymers into Biogels for Regenerative Wound Healing and Versatile Skin Bioelectronics.

An increasing utilization of wound-related therapeutic materials and skin bioelectronics urges the development of multifunctional biogels for personal therapy and health management. Nevertheless, conventional dressings and skin bioelectronics with single function, mechanical mismatches, and impracticality severely limit their widespread applications in clinical. Herein, we explore a gelling mechanism, fabrication method, and functionalization for broadly applicable food biopolymers-based biogels that unite the challenging needs of elastic yet injectable wound dressing and skin bioelectronics in a single system. We combine our biogels with functional nanomaterials, such as cuttlefish ink nanoparticles and silver nanowires, to endow the biogels with reactive oxygen species scavenging capacity and electrical conductivity, and finally realized the improvement in diabetic wound microenvironment and the monitoring of electrophysiological signals on skin. This line of research work sheds light on preparing food biopolymers-based biogels with multifunctional integration of wound treatment and smart medical treatment.

High-Performance Extended-Gate Field-Effect Transistor for Kinase Sensing in Aß Accumulation of Alzheimer's Disease.

An amyloid-beta peptide (Aß) is generally believed to be a pathological marker of Alzheimer's disease (AD), but it is still of great significance to explore the upstream and downstream relationship of Aß in AD. It is previously reported that c-Abl, a nonreceptor tyrosine kinase, can be activated by Aß, but the interaction between Aß and c-Abl is still unknown. Herein, an extended-gate field-effect transistor (EG-FET)-based sensor has been developed to monitor the level of c-Abl with high sensitivity and selectivity. Our peptide-functionalized EG-FET sensor as the signal transducer can follow c-Abl activity with electron transfer by its specific phosphorylation. The sensor presents a good linear correlation over c-Abl concentrations of 1 pg/mL to 3.05 µg/mL. The sensor was successfully applied to quantify c-Abl activity in the brain tissue of AD transgenic mice, and the interaction between c-Abl and Aß in AD mice was explored by administering the c-Abl inhibitor (imatinib) and the agonist (DPH). Our work is expected to provide an important reference for early diagnosis and treatment of AD.

Electrochemical Strategy for Analyzing the Co-evolution of Cu2+ and •OH Levels at the Early Stages of Transgenic AD Mice.

As more researchers have acknowledged that the aggregation of amyloid ß (Aß) peptides might only be a pathological phenomenon that appears during the course of Alzheimer's disease (AD), it is therefore of great significance to have a preclinical or an early clinical diagnosis. Cu2+ dyshomeostasis and oxidative stress, such as hydroxyl radical (•OH), are found to be associated with peptide aggregations. However, we still do not know how the levels of Cu2+ and •OH are altered in the brain before massive Aß plaques appear. Herein, we demonstrated the design and application of a sensitive electrochemical sensor to monitor Cu2+ and •OH simultaneously in one system without obvious cross-talk. The electrode was fabricated using black phosphorus-loaded Au (BP-Au) nanoparticles, which were then sequentially linked with DNA1, DNA2-labeled Au (Au-DNA2) nanoparticles, and methylene blue (MB). Cu2+ was first recognized and captured onto the sensor by BP with high selectivity and then produced a reduction current at around -0.01 V. The •OH quantification was established on the cleavage of the hybrid structure between DNA1 and BP-Au upon the appearance of •OH in the phosphate-buffered saline (PBS), leading to the depletion of the voltammetric response of MB around -0.25 V. Good linear correlations were obtained over concentrations of 0.5-127.5 µM for Cu2+ and 0.5-96.0 µM for •OH. Most importantly, the developed sensor was successfully applied to track the variations of the two species in brain tissues from APP/PS1 transgenic AD mice at the early stages before massive Aß plaques appeared.

In vivo monitoring of superoxide anion from Alzheimer's rat brains with functionalized ionic liquid polymer decorated microsensor.

The superoxide anion (O2•-) is an important reactive oxygen species (ROS) in the brain system, which has been associated with the development of many neurological diseases, including Alzheimer's disease (AD). Herein, we introduced a carbon fiber microelectrode (CFME) based in vivo technique for specific and sensitive monitoring of the O2•- radical in the living brains of both normal and AD model rats. Compared with other reported superoxide dismutase (SOD) electrochemical biosensors, the microsensor presented in our work was featured in the coating of a functionalized ionic liquid polymer (PIL) onto PB nanoparticles (PBNPs) and carbon nanotubes (CNT). It was demonstrated that the cationic and carboxyl-rich PILs provided abundant interaction sites with SOD to prevent enzyme leakage from sensor, which was beneficial for the enhancement of sensitivity. Additionally, CCK-8 assay and autoxidation of pyrogallol tests showed that MCF-7 cells maintained a high viability after incubated with PIL and most of the SOD bioactivity was retained in the presence of PIL, which implied the PIL itself possessed an excellent biocompatibility. These properties allow the sensor to track the fluctuation of O2•- levels in vivo between normal and AD rats. This is the first report on application of functionalized PIL to reveal the O2•- related pathological process of AD.

Simultaneous Monitoring of Amyloid-ß (Aß) Oligomers and Fibrils for Effectively Evaluating the Dynamic Process of Aß Aggregation.

Herein, we provide a proof of concept for a novel strategy that targets the assessment of the aggregation of amyloid-ß (Aß) by simultaneously determining its oligomers (Aßo) and fibrils (Aßf) in one analytical system. By fabricating and combining two immunosensors for Aßo and Aßf, respectively, we constructed a two-channel electrochemical system. The ratio of Aßf to Aßo was calculated and taken as a possible criterion for evaluating the extent of aggregation. Thereby, the presence of and transformation between oligomers and fibrils were accurately probed by incubating the Aß monomer for different times and then calculating the ratios of Aßf to Aßo. The applicability of this method was further validated by tracking the dynamic progress of Aß aggregation in the cerebrospinal fluid and tissues of Alzheimer's disease (AD) rats, which revealed that the ratio of Aßf to Aßo in rat brain gradually increased with the progression of AD, which was indicative of the severity of peptide aggregation during this process. Overall, this study represents the first example of a quantitative strategy for precisely evaluating the aggregation process that is related to pathological events in AD brain.

Transition metal-coordinated graphitic carbon nitride dots as a sensitive and facile fluorescent probe for ß-amyloid peptide detection.

Herein, we developed a sensitive graphitic carbon nitride quantum dot (gCNQD)-based fluorescent strategy for ß-amyloid peptide monomer (Aß) determination down to the ng mL-1 level for the first time. To realize this goal, the nanostructured gCNQDs were firstly coordinated with four transition metal ions (Cu2+, Cu+, Fe3+, Zn2+). Our findings showed that the fluorescence (FL) intensity of gCNQDs was quenched in the presence of these metal ions possibly due to the effective chelation with the nitrogen element in gCNQDs and subsequent photoinduced electron transfer (PET) of gCNQDs. The degree of fluorescence quenching was found to be the most intense with the addition of Cu2+ and therefore, we selected Cu2+ as the quencher for the following Aß determination. Through binding to Cu2+, the introduction of Aß unexpectedly induced a further decline of FL intensity. Importantly, on account of different peptide sequences coexisting in the same cerebral system, including Aß1-11, Aß1-16, Aß1-38, Aß1-40 and Aß1-42, their affinities to Cu2+ could be reflected by the distinguished declining extent of FL intensity. The possible mechanism of Aß sensing by the probe was clarified by TEM characterization. The developed fluorescent biosensor was demonstrated to give a wide linear range from 1 to 700 ng mL-1 and a low detection limit of 0.18 ng mL-1 for Aß1-42. In the end, the proposed fluorescence approach was successfully applied to monitoring of Aß1-42 variations in the cortex and hippocampus of AD rats.

Combined determination of copper ions and ß-amyloid peptide by a single ratiometric electrochemical biosensor.

Copper ions (Cu2+) play a critical role in biological processes and are directly involved in ß-amyloid peptide (Aß) aggregation, which is responsible for the occurrence and development of Alzheimer's disease (AD). Therefore, combined determination of Cu2+ and Aß in one analytical system is of great significance to understand the exact nature of the AD event. This work presents a novel ratiometric electrochemical biosensor for the dual determination of Cu2+ and Aß1-42. This unique sensor is based on a 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS) and poly(diallyldimethylammonium chloride) (PDDA)-bi functionalized single-walled carbon nanotubes (ABTS-PDDA/CNTs) composite. The inclusion of ABTS not only enhanced the sensitivity, but it also acted as an inner reference molecule to improve detection accuracy. The specific recognition of Cu2+ was realized by neurokinin B (NKB) coatings on the ABTS-PDDA/CNTs surface to form a [CuII(NKB)2] complex with Cu2+. The ABTS-PDDA/CNTs-NKB modified electrode also displayed an excellent electrochemical response toward the Aß1-42 monomer, when a certain amount of the Aß1-42 monomer was added to Cu2+-contained PBS buffer, which was due to the release of Cu2+ from the [CuII(NKB)2] complex through Aß binding to Cu2+. Meanwhile, our work showed that Cu2+ bound Aß1-42 was concentration-dependent. Consequently, the presented electrochemical approach was capable of quantifying two important biological species associated with AD by one single biosensor, with the detection limits of 0.04 µM for Cu2+ and 0.5 ng mL-1 for Aß1-42, respectively. Finally, the ratiometric electrode was successfully applied for monitoring Cu2+ and Aß1-42 variations in plasma and hippocampus of normal and AD rats.

Quantification of liver fibrosis via second harmonic imaging of the Glisson's capsule from liver surface.

Liver surface is covered by a collagenous layer called the Glisson's capsule. The structure of the Glisson's capsule is barely seen in the biopsy samples for histology assessment, thus the changes of the collagen network from the Glisson's capsule during the liver disease progression are not well studied. In this report, we investigated whether non-linear optical imaging of the Glisson's capsule at liver surface would yield sufficient information to allow quantitative staging of liver fibrosis. In contrast to conventional tissue sections whereby tissues are cut perpendicular to the liver surface and interior information from the liver biopsy samples were used, we have established a capsule index based on significant parameters extracted from the second harmonic generation (SHG) microscopy images of capsule collagen from anterior surface of rat livers. Thioacetamide (TAA) induced liver fibrosis animal models was used in this study. The capsule index is capable of differentiating different fibrosis stages, with area under receiver operating characteristics curve (AUC) up to 0.91, making it possible to quantitatively stage liver fibrosis via liver surface imaging potentially with endomicroscopy.

Reassignment of scattered emission photons in multifocal multiphoton microscopy.

Multifocal multiphoton microscopy (MMM) achieves fast imaging by simultaneously scanning multiple foci across different regions of specimen. The use of imaging detectors in MMM, such as CCD or CMOS, results in degradation of image signal-to-noise-ratio (SNR) due to the scattering of emitted photons. SNR can be partly recovered using multianode photomultiplier tubes (MAPMT). In this design, however, emission photons scattered to neighbor anodes are encoded by the foci scan location resulting in ghost images. The crosstalk between different anodes is currently measured a priori, which is cumbersome as it depends specimen properties. Here, we present the photon reassignment method for MMM, established based on the maximum likelihood (ML) estimation, for quantification of crosstalk between the anodes of MAPMT without a priori measurement. The method provides the reassignment of the photons generated by the ghost images to the original spatial location thus increases the SNR of the final reconstructed image.

Experimenting liver fibrosis diagnostic by two photon excitation microscopy and Bag-of-Features image classification.

The accurate staging of liver fibrosis is of paramount importance to determine the state of disease progression, therapy responses, and to optimize disease treatment strategies. Non-linear optical microscopy techniques such as two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) can image the endogenous signals of tissue structures and can be used for fibrosis assessment on non-stained tissue samples. While image analysis of collagen in SHG images was consistently addressed until now, cellular and tissue information included in TPEF images, such as inflammatory and hepatic cell damage, equally important as collagen deposition imaged by SHG, remain poorly exploited to date. We address this situation by experimenting liver fibrosis quantification and scoring using a combined approach based on TPEF liver surface imaging on a Thioacetamide-induced rat model and a gradient based Bag-of-Features (BoF) image classification strategy. We report the assessed performance results and discuss the influence of specific BoF parameters to the performance of the fibrosis scoring framework.

qFibrosis: a fully-quantitative innovative method incorporating histological features to facilitate accurate fibrosis scoring in animal model and chronic hepatitis B patients.

BACKGROUND & AIMS: There is increasing need for accurate assessment of liver fibrosis/cirrhosis. We aimed to develop qFibrosis, a fully-automated assessment method combining quantification of histopathological architectural features, to address unmet needs in core biopsy evaluation of fibrosis in chronic hepatitis B (CHB) patients. METHODS: qFibrosis was established as a combined index based on 87 parameters of architectural features. Images acquired from 25 Thioacetamide-treated rat samples and 162 CHB core biopsies were used to train and test qFibrosis and to demonstrate its reproducibility. qFibrosis scoring was analyzed employing Metavir and Ishak fibrosis staging as standard references, and collagen proportionate area (CPA) measurement for comparison. RESULTS: qFibrosis faithfully and reliably recapitulates Metavir fibrosis scores, as it can identify differences between all stages in both animal samples (p<0.001) and human biopsies (p<0.05). It is robust to sampling size, allowing for discrimination of different stages in samples of different sizes (area under the curve (AUC): 0.93-0.99 for animal samples: 1-16 mm(2); AUC: 0.84-0.97 for biopsies: 10-44 mm in length). qFibrosis can significantly predict staging underestimation in suboptimal biopsies (<15 mm) and under- and over-scoring by different pathologists (p<0.001). qFibrosis can also differentiate between Ishak stages 5 and 6 (AUC: 0.73, p=0.008), suggesting the possibility of monitoring intra-stage cirrhosis changes. Best of all, qFibrosis demonstrates superior performance to CPA on all counts. CONCLUSIONS: qFibrosis can improve fibrosis scoring accuracy and throughput, thus allowing for reproducible and reliable analysis of efficacies of anti-fibrotic therapies in clinical research and practice.

Hepatic stellate cell-targeted delivery of hepatocyte growth factor transgene via bile duct infusion enhances its expression at fibrotic foci to regress dimethylnitrosamine-induced liver fibrosis.

Liver fibrosis generates fibrotic foci with abundant activated hepatic stellate cells and excessive collagen deposition juxtaposed with healthy regions. Targeted delivery of antifibrotic therapeutics to hepatic stellate cells (HSCs) might improve treatment outcomes and reduce adverse effects on healthy tissue. We delivered the hepatocyte growth factor (HGF) gene specifically to activated hepatic stellate cells in fibrotic liver using vitamin A-coupled liposomes by retrograde intrabiliary infusion to bypass capillarized hepatic sinusoids. The antifibrotic effects of DsRed2-HGF vector encapsulated within vitamin A-coupled liposomes were validated by decreases in fibrotic markers in vitro. Fibrotic cultures transfected with the targeted transgene showed a significant decrease in fibrotic markers such as transforming growth factor-ß1. In rats, dimethylnitrosamine-induced liver fibrosis is manifested by an increase in collagen deposition and severe defenestration of sinusoidal endothelial cells. The HSC-targeted transgene, administered via retrograde intrabiliary infusion in fibrotic rats, successfully reduced liver fibrosis markers alpha-smooth muscle actin and collagen, accompanied by an increase in the expression of DsRed2-HGF near the fibrotic foci. Thus, targeted delivery of HGF gene to hepatic stellate cells increased the transgene expression at the fibrotic foci and strongly enhanced its antifibrotic effects.

Toward surface quantification of liver fibrosis progression.

Monitoring liver fibrosis progression by liver biopsy is important for certain treatment decisions, but repeated biopsy is invasive. We envision redefinition or elimination of liver biopsy with surface scanning of the liver with minimally invasive optical methods. This would be possible only if the information contained on or near liver surfaces accurately reflects the liver fibrosis progression in the liver interior. In our study, we acquired the second-harmonic generation and two-photon excitation fluorescence microscopy images of liver tissues from bile duct-ligated rat model of liver fibrosis. We extracted morphology-based features, such as total collagen, collagen in bile duct areas, bile duct proliferation, and areas occupied by remnant hepatocytes, and defined the capsule and subcapsular regions on the liver surface based on image analysis of features. We discovered a strong correlation between the liver fibrosis progression on the anterior surface and interior in both liver lobes, where biopsy is typically obtained. The posterior surface exhibits less correlation with the rest of the liver. Therefore, scanning the anterior liver surface would obtain similar information to that obtained from biopsy for monitoring liver fibrosis progression.