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An amyloid-beta peptide (Aβ) 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 Aβ in AD. It is previously reported that c-Abl, a nonreceptor tyrosine kinase, can be activated by Aβ, but the interaction between Aβ 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 Aβ 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.

As more researchers have acknowledged that the aggregation of amyloid β (Aβ) 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. Cu

Herein, we provide a proof of concept for a novel strategy that targets the assessment of the aggregation of amyloid-β (Aβ) by simultaneously determining its oligomers (Aβo) and fibrils (Aβf) in one analytical system. By fabricating and combining two immunosensors for Aβo and Aβf, respectively, we constructed a two-channel electrochemical system. The ratio of Aβf to Aβo 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 Aβ monomer for different times and then calculating the ratios of Aβf to Aβo. The applicability of this method was further validated by tracking the dynamic progress of Aβ aggregation in the cerebrospinal fluid and tissues of Alzheimer's disease (AD) rats, which revealed that the ratio of Aβf to Aβo 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.

Herein, we developed a sensitive graphitic carbon nitride quantum dot (gCNQD)-based fluorescent strategy for β-amyloid peptide monomer (Aβ) 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 Aβ determination. Through binding to Cu2+, the introduction of Aβ unexpectedly induced a further decline of FL intensity. Importantly, on account of different peptide sequences coexisting in the same cerebral system, including Aβ1-11, Aβ1-16, Aβ1-38, Aβ1-40 and Aβ1-42, their affinities to Cu2+ could be reflected by the distinguished declining extent of FL intensity. The possible mechanism of Aβ 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 Aβ1-42. In the end, the proposed fluorescence approach was successfully applied to monitoring of Aβ1-42 variations in the cortex and hippocampus of AD rats.

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.

Blood oxygen saturation (SO2) reflects the oxygenation level in blood transport and tissue. Previous studies have shown the capability of non-invasive quantitative measurements of SO2 by multi-wavelength photoacoustic (PA) spectroscopy for diagnosis of brain, tumor hemodynamics, and other pathophysiological phenomena. Here, we report a newly proposed method by combining PA and scattered light signals wherein imposing a hypothesis that scattering intensity is linear to the concentrations of oxygenated hemoglobin and deoxygenated hemoglobin weighed by blood scattering coefficients. A rigorous theoretical relationship between PA and scattering signals is thus established, making it possible that SO2 can be measured with only one excitation wavelength after calibration. To verify the theory basis, both dual-ink phantoms and fresh porcine blood sample have been employed in the experiments. The phantom experiment is able to quantify the concentration of mixed red–green ink that is in precise agreement with pre-set values. The in vitro experiment with fresh porcine blood was conducted, and the results of the proposed single-wavelength method achieved reasonable accuracy with 5%–8% errors. These demonstrated that the proposed single-wavelength SO2 detection method is able to provide non-invasive, accurate measurement of blood oxygenation combining optical absorption and scattering.

Copper ions (Cu2+) play a critical role in biological processes and are directly involved in β-amyloid peptide (Aβ) aggregation, which is responsible for the occurrence and development of Alzheimer's disease (AD). Therefore, combined determination of Cu2+ and Aβ 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 Aβ1–42. 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 Aβ1–42 monomer, when a certain amount of the Aβ1–42 monomer was added to Cu2+-contained PBS buffer, which was due to the release of Cu2+ from the [CuII(NKB)2] complex through Aβ binding to Cu2+. Meanwhile, our work showed that Cu2+ bound Aβ1–42 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 Aβ1–42, respectively. Finally, the ratiometric electrode was successfully applied for monitoring Cu2+ and Aβ1–42 variations in plasma and hippocampus of normal and AD rats.

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

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.

Blood oxygen saturation (SO2) reflects the oxygenation level in blood transport and tissue. Previous studies have shown the capability of non-invasive quantitative measurements of SO2 by multi-wavelength photoacoustic (PA) spectroscopy for diagnosis of brain, tumor hemodynamics and other pathophysiological phenomena. However, those multi-wavelength methods require a tunable laser or multiple lasers which are relatively expensive and bulky for filed measurement environment and applications. Besides, the operation of multiple wavelengths, calibration procedures and data processing gets system complicated, which reduces the feasibility and flexibility for continuous real-time monitoring. Here we report a newly proposed method by combining PA and scattered light signals wherein imposing a hypothesis that scattering intensity is linear to the concentrations of oxygenated hemoglobin and deoxygenated hemoglobin weighed by blood scattering coefficients. A rigorous theoretical relationship between PA and scattering signals is thus established, making it possible that SO2 can be measured with only one excitation wavelength. To verify the theory basis, both dual-ink phantoms and fresh porcine blood sample have been employed in the experiments. The phantom experiment is able to quantify the concentration of mixed red-green ink that is in precise agreement with pre-set values. The ex vivo experiment with fresh porcine blood was conducted and the results of the proposed single-wavelength method achieved high accuracy of 1% - 4% errors. These demonstrated that the proposed single-wavelength SO2 detection is able to provide non-invasive, accurate measurement of blood oxygenation, and herein create potential for applying it to real clinical applications with low cost and high flexibility. Published version

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., RO1 EY017656, Singapore-MIT Alliance for Research and Technology, NIH P41EB015871, 5 R01 NS051320, 4R44EB012415, NSF CBET-0939511, MIT Skoltech Initiative, David H. Koch Institute for Integrative Cancer Research at MIT

A bipyridine centered donor–acceptor–donor (D–π–A–π–D) type ratiometric fluorescent molecular probe exhibited an unprecedented enhancement in the two-photon absorption (2PA) cross section upon Zn2+ binding. Moreover, owing to the excited state charge-transfer of the fluorophore π-backbone, a significant enhancement in the two-photon (2P) excited fluorescence intensity was observed upon Zn2+ binding, resulting in a 13-fold enhancement in the 2PA cross section and a 9-fold enhancement in fluorescence brightness at 620 nm when compared to the cation-free fluorophore. The large 2PA cross section of 1433 GM and 2P action cross section (860 GM), with an excellent 2P excited fluorescence variation from 517 to 620 nm upon Zn2+ binding, facilitated the ratiometric monitoring of free zinc ions in cells. The low cytotoxicity and good photostability of the fluorophore allowed two-photon Zn2+ imaging of HeLa cells. In addition, in vivo two-photon imaging of Zn2+ ions in hepatocytes of live rats illustrated the viability of the probe in tissue imaging and monitoring of free zinc ions in live cells. NRF (Natl Research Foundation, S’pore) Accepted version

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., Romania. Executive Agency for Higher Education, Research, Development and Innovation Funding (research grant PN-II-PT-PCCA-2011-3.2-1162), Rectors' Conference of the Swiss Universities (SCIEX NMS-CH research fellowship nr. 12.135), Singapore. Agency for Science, Technology and Research (R-185-000-182-592), Singapore. Biomedical Research Council, Institute of Bioengineering and Nanotechnology (Singapore), Singapore-MIT Alliance (Computational and Systems Biology Flagship Project funding (C-382-641-001-091)), Singapore-MIT Alliance for Research and Technology (SMART BioSyM and Mechanobiology Institute of Singapore (R-714-001-003-271))

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-β1. 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.

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.

The correlation of forward second harmonic generation (SHG) signal and backward SHG signal in different liver fibrosis stages was investigated. We found that three features, including the collagen percentage for forward SHG, the collagen percentage for backward SHG, and the average intensity ratio of two kinds of SHG signals, can quantitatively stage liver fibrosis in thioacetamide-induced rat model. We demonstrated that the combination of all three features by using a support vector machine classification algorithm can provide a more accurate prediction than each feature alone in fibrosis diagnosis.

Various structural features on the liver surface reflect functional changes in the liver. The visualization of these surface features with molecular specificity is of particular relevance to understanding the physiology and diseases of the liver. Using multi-photon microscopy (MPM), we have developed a label-free, three-dimensional quantitative and sensitive method to visualize various structural features of liver surface in living rat. MPM could quantitatively image the microstructural features of liver surface with respect to the sinuosity of collagen fiber, the elastic fiber structure, the ratio between elastin and collagen, collagen content, and the metabolic state of the hepatocytes that are correlative with the pathophysiologically induced changes in the regions of interest. This study highlights the potential of this technique as a useful tool for pathophysiological studies and possible diagnosis of the liver diseases with further development.