Spatially controlled diffusion range of tumor-associated angiogenic factors to develop a tumor model using a microfluidic resistive circuit.

Developing a tumor model with vessels has been a challenge in microfluidics. This difficulty is because cancer cells can overgrow in a co-culture system. The up-regulation of anti-angiogenic factors during the initial tumor development can hinder neovascularization. The standard method is to develop a quiescent vessel network before loading a tumor construct in an adjacent chamber, which simulates the interaction between a tumor and its surrounding vessels. Here, we present a new method that allows a vessel network and a tumor to develop simultaneously in two linked chambers. The physiological environment of these two chambers is controlled by a microfluidic resistive circuit using two symmetric long microchannels. Applying the resistive circuit, a diffusion-dominated environment with a small 2-D pressure gradient is created across the two chambers with velocity <10.9 nm s-1 and Péclet number <6.3 × 10-5. This 2-D pressure gradient creates a V-shaped velocity clamp to confine the tumor-associated angiogenic factors at pores between the two chambers, and it has two functions. At the early stage, vasculogenesis is stimulated to grow a vessel network in the vessel chamber with minimal influence from the tumor that is still developed in the adjacent chamber. At the post-tumor-development stage, the induced steep concentration gradient at pores mimics vessel-tumor interactions to stimulate angiogenesis to grow vessels toward the tumor. Applying this method, we demonstrate that vasculogenic vessels can grow first, followed by stimulating angiogenesis. Angiogenic vessels can grow into stroma tissue up to 1.3 mm long, and vessels can also grow into or wrap around a 625 µm tumor spheroid or a tumor tissue developed from a cell suspension. In summary, our study suggests that the interactions between a developing vasculature and a growing tumor must be controlled differently throughout the tissue development process, including at the early stage when vessels are still forming and at the later stage when the tumor needs to interact with the vessels.

[Detection and analysis of moving reaction boundary-based electrophoresis distance using smartphone images].

Electrophoresis titration (ET) based on the moving reaction boundary (MRB) theory can detect the analyte contents in different samples by converting content signals into distance signals. However, this technique is only suitable for on-site qualitative testing, and accurate quantification relies on complex optical equipment and computers. Hence, applying this method to real-time point-of-care testing (POCT) is challenging. In this study, we developed a smartphone-based ET system based on a visual technique to achieve real-time quantitative detection. First, we developed a portable quantitative ET device that can connect to a smartphone; this device consisted of five components, namely, an ET chip, a power module, a microcontroller, a liquid crystal display screen, and a Bluetooth module. The device measured 10 cm×15 cm×2.5 cm, weighed 300 g, and was easy to hold. Thus, it is suitable for on-site testing with a run time of only 2-4 min. An assistant mobile software program was also developed to control the device and perform ET. The colored electrophoresis boundary can be captured using the smartphone camera, and quantitative detection results can be obtained in real time. Second, we proposed a quantitative algorithm based on ET channels. The software was used to recognize the boundary migration distance of three channels, a standard curve based on two given contents of the standards was established using the two-point method, and the content of the test sample was calculated. Human serum albumin (HSA) and uric acid (UA) were used as a model protein and biosample, respectively, to test the performance of the detection system. For HSA detection, different HSA solutions were mixed with a polyacrylamide gel (PAG) stock solution, phenolphthalein was added as an indicator, and sodium persulfate and tetramethyl ethylenediamine (TEMED) were used to promote polymerization to form a gel. For UA detection, agarose gel was filled into the ET channel, the UA sample, urate oxidase, and leucomalachite green were added into the anode cell and incubated for 20 min. ET was then performed. The fitting goodness (R2) values of HSA and UA were 0.9959 and 0.9935, respectively, with a linear range of 0.5-35.0 g/L and a log-linear range of 100-4000 µmol/L. The limits of detection for HSA and UA were 0.05 g/L and 50 µmol/L, respectively, and the corresponding relative standard deviations (RSDs) were not greater than 2.87% and 3.21%, respectively. These results demonstrate that the detection system has good accuracy and sensitivity. Clinical samples collected from healthy volunteers were used as target blood samples, and the developed system was used to measure serum total protein and UA levels. Serum samples from five volunteers were selected, standard curves of total serum protein and UA were established, and the test results were compared with hospital standard testing results. The relative errors for serum total protein and UA were less than 6.03% and 6.21%, respectively, and the corresponding RSDs were less than 3.72% and 5.84%, respectively. These findings verify the accuracy and reliability of the proposed detection system. The smartphone-based ET detection system introduced in this paper presents several advantages. First, it enables the portable real-time detection of total serum protein and UA. Second, compared with traditional ET strategies based on colored boundaries, it does not rely on optical detection equipment or computers to obtain quantitative detection results; as such, it can reduce the complexity of the operation and provide portability and real-time metrics. Third, the detection of two biomarkers, serum total protein and UA, is achieved on the same device, thereby improving the multitarget detection potential of the ET method. These advantages render the developed method a promising detection platform for clinical applications and real-time POCT.

Metastasis and immunosuppression promoted by mtDNA and PD-L1 in extracellular vesicles are reversed by WGP ß-glucan in oral squamous cell carcinoma.

The suppressive regulatory T cells (Treg) are frequently upregulated in cancer patients. This study aims to demonstrate the hypothesis that arecoline could induce the secretion of mitochondrial (mt) DNA D-loop and programmed cell death-ligand 1 (PD-L1) in extracellular vesicles (EVs), and attenuate T-cell immunity by upregulated Treg cell numbers. However, the immunosuppression could be reversed by whole glucan particle (WGP) ß-glucan in oral squamous cell (OSCC) patients. Arecoline-induced reactive oxygen specimen (ROS) production and cytosolic mtDNA D-loop were analyzed in OSCC cell lines. mtDNA D-loop, PD-L1, IFN-γ, and Treg cells were also identified for the surgical specimens and sera of 60 OSCC patients. We demonstrated that higher mtDNA D-loop, PD-L1, and Treg cell numbers were significantly correlated with larger tumor size, nodal metastasis, advanced clinical stage, and areca quid chewing. Furthermore, multivariate analysis confirmed that higher mtDNA D-loop levels and Treg cell numbers were unfavorable independent factors for survival. Arecoline significantly induced cytosolic mtDNA D-loop leakage and PD-L1 expression, which were packaged by EVs to promote immunosuppressive Treg cell numbers. However, WGP ß-glucan could elevate CD4+ and CD8+ T-cell numbers, mitigate Treg cell numbers, and promote oral cancer cell apoptosis. To sum up, arecoline induces EV production carrying mtDNA D-loop and PD-L1, and in turn elicits immune suppression. However, WGP ß-glucan potentially enhances dual effects on T-cell immunity and cell apoptosis and we highly recommend its integration with targeted and immune therapies against OSCC.

[Determination of human serum total protein via electrophoresis titration and capacitively coupled contactless conductivity detection].

Serum total protein refers to the sum of all proteins in the serum, and its content determination is relevant to human health monitoring and disease diagnosis. However, existing detection techniques present a number of limitations; for example, the Kjeldahl method suffers from the negative effects of interfering substances such as non-protein nitrogen (NPN). Although the electrophoresis titration (ET) method has solved interference problems to some extent, the current ET technique relies on optical detection methods, which increases the tediousness of the operation. This study addresses the challenge of accurate serum total protein detection by combining the traditional ET technique with capacitively coupled contactless conductivity detection (C4D). The research contributions of this work are multifold. First, it presents the first development of an ET-C4D detection system, which consists of six components: an ET power module, an ET chip, a C4D sensing module, a detection module, a data acquisition card, and software. The developed system can capture the conductivity of substances in the channel using the software developed by our laboratory during ET. The detection system can be used to quantify the total protein content in human serum without the addition of specific labeling reagents or using optical detection equipment, and its running time is approximately 300 s. Second, this research proposes the corresponding principle of the system. Under an electric field, ion migration results in different pH levels before and after the boundary, leading to a protein surface charge difference. The maintenance of the electrical neutrality of the substances in the detection channel is related to the protein surface charge; therefore, the ion concentration distribution of the substances in the detection channel changes as the protein surface charge varies. A plot of conductivity as a function of running time showed an "inverted clock shape", first falling and then rising. Owing to the addition of different types and concentrations of proteins, the microenvironment of the entire system changes, resulting in different changes in conductivity. Third, the performance of the detection system was tested using human serum albumin (HSA) standard protein, which was mixed with polyacrylamide gel (PAG) mother liquor, riboflavin, etc., and irradiated under ultraviolet light for 10 min to form a gel. The ET experiments were then carried out. The shape of the conductivity curve was consistent with the proposed principle, and the higher the HSA concentration, the lower the conductivity curve trough, followed by a lagged time of the trough. Quantitative analysis of the conductivity signals showed that the linear range was 0.25-3.00 g/L, with a linearity of up to 0.98. The limit of detection (LOD) was 0.01 g/L, the relative standard deviation (RSD) was 1.90%, and the relative error of the test values was <7.20%, indicating the good detection stability and sensitivity of the system. Clinical samples collected from healthy volunteers were used as target blood samples for serum total protein content measurement using our detection system. Blood samples from a volunteer were used to obtain a standard curve, and the serum samples of other four volunteers were selected for ET-C4D and biuret detection. The results showed that the relative errors between the two methods were within 4.43%, indicating the accuracy and reliability of the detection system. The advantages of the ET-C4D detection system proposed in this paper are as follows: (i) ET-C4D realizes the rapid detection of total serum protein content based on the ET technique; (ii) compared with the traditional protein ET technique, the ET-C4D method does not rely on specific labeling components or optical detection equipment, thereby reducing the complexity of the operation; and (iii) the output signal of ET-C4D can be used for quantitative analysis with excellent analytical performance and high accuracy. These merits highlight the potential of the developed system for clinical application and biochemical analysis.

Review on the Effects of Electrochemical Exfoliation Parameters on the Yield of Graphene Oxide.

Recent years have witnessed many breakthroughs in research on graphene as well as a significant improvement in the electrochemical synthesis methods of graphene oxide (GO). GO is a derivative of graphene which has attracted the focus of worldwide scientists and researchers because of its hydrophilic and easily functionalized properties. The electrochemical approach is popular because it saves time, creates zero explosion risk, releases no hazardous gases, and avoids environmental pollution. Although recent publications show that the green, rapid, and mass electrochemical synthesis of GO has more advantages as compared with the traditional Hummers method, it is crucial to study the effects of reaction parameters. Herein, we review recent various works regarding the influences of various reaction parameters on the synthesis of GO sheets. The advancement, current challenges, and solutions of electrochemical synthesis methods of GO are also outlined. Through this review, we hope to spark some clear ideas for anyone who wants to scale up the yield of GO.

Postoperative survival of extrahepatic and intrahepatic cholangiocarcinoma after surgery: a population-based cohort.

OBJECTIVES: The study was designed to clarify the difference between extrahepatic cholangiocarcinoma (ECC) and intrahepatic cholangiocarcinoma (ICC) in postoperative cancer-specific death. DESIGN: Patients diagnosed with ECC and ICC after surgery, who are identified from the Surveillance, Epidemiology and End Results programme, are eligible for this retrospective cohort study. SETTING: Survival between groups was compared using the traditional Kaplan-Meier method and the cumulative incidence function (CIF) method. Propensity score-matched (PSM) analysis was conducted to balance the differences in vital variables between groups. The HR and 95% CI for ECC relative to ICC were used to quantify the risk of death. Subgroup analysis was further used to evaluate the stability of the differences between groups. RESULTS: The study included 876 patients with ECC and 1194 patients with ICC. Before PSM, with the Kaplan-Meier method, postoperative overall survival and cancer-specific death for ECC were worse than those for ICC. However, with the CIF method, no difference in postoperative cancer-specific death was found. After PSM, all differences in the considered traits were balanced, and 173 pairs of patients were retained. Survival analysis found that there was no difference in postoperative all-cause death (Kaplan-Meier method, p=0.186) or cancer-specific death (Kaplan-Meier and CIF methods, p=0.500 and p=0.913, respectively), which was consistent with subgroup analysis. CONCLUSIONS: ECC and ICC showed no difference in postoperative cancer-specific death, both in the natural state and in multiple variable-matched conditions. TRIAL REGISTRATION NUMBER: researchregistry4175.

Longitudinal shear wave elasticity measurements of millimeter-sized biomaterials using a single-element transducer platform.

Temporal variations of the extracellular matrix (ECM) stiffness profoundly impact cellular behaviors, possibly more significantly than the influence of static stiffness. Three-dimensional (3D) cell cultures with tunable matrix stiffness have been utilized to characterize the mechanobiological interactions of elasticity-mediated cellular behaviors. Conventional studies usually perform static interrogations of elasticity at micro-scale resolution. While such studies are essential for investigations of cellular mechanotransduction, few tools are available for depicting the temporal dynamics of the stiffness of the cellular environment, especially for optically turbid millimeter-sized biomaterials. We present a single-element transducer shear wave (SW) elasticity imaging system that is applied to a millimeter-sized, ECM-based cell-laden hydrogel. The single-element ultrasound transducer is used both to generate SWs and to detect their arrival times after being reflected from the side boundaries of the sample. The sample's shear wave speed (SWS) is calculated by applying a time-of-flight algorithm to the reflected SWs. We use this noninvasive and technically straightforward approach to demonstrate that exposing 3D cancer cell cultures to X-ray irradiation induces a temporal change in the SWS. The proposed platform is appropriate for investigating in vitro how a group of cells remodels their surrounding matrix and how changes to their mechanical properties could affect the embedded cells in optically turbid millimeter-sized biomaterials.

Sonoporation based on repeated vaporization of gold nanodroplets.

BACKGROUND: Gold nanodroplets (AuNDs) have been proposed as agents for photothermal therapy and photoacoustic imaging. Previously, we demonstrated that the sonoporation can be more effectively achieved with synchronized optical and acoustic droplet vaporization. By applying a laser pulse at the rarefactional phase of the ultrasound (US) pulse, the vaporization threshold can be reached at a considerably lower laser average power. However, a large loading quantity of the AuNDs may increase the risk of air embolism. The destruction of phase-shifted AuNDs at the inertial cavitation stage leads to a reduced drug delivery performance. And it also causes instability of echogenicity during therapeutic monitoring. PURPOSE: In this study, we propose to further improve the sonoporation effectiveness with repeated vaporization. In other words, the AuNDs repeatedly undergo vaporization and recondensation so that sonoporation effects are accumulated over time at lower energy requirements. Previously, repeated vaporization has been demonstrated as an imaging contrast agent. In this study, we aim to adopt this repeated vaporization scheme for sonoporation. METHODS: Perfluoropentane NDs with a shell made of human serum albumin were used as the US contrast agents. Laser pulses at 808 nm and US pulses of 1 MHz were delivered for triggering vaporization and inertial cavitation of NDs. We detected the vaporization and cavitation effects under different activation firings, US peak negative pressures (PNPs), and laser fluences using 5- and 10-MHz focused US receivers. Numbers of calcein-AM and propidium iodide signals uptake by BNL hepatocarcinoma cancer cells were used to evaluate the sonoporation and cell death rate of the cells. RESULTS: We demonstrate that sonoporation can be realized based on repeatable vaporization instead of the commonly adopted inertial cavitation effects. In addition, it is found that the laser fluence and the acoustic pressure can be reduced. As an example, we demonstrate that the acoustic and optical energy for achieving a similar level of sonoporation rate can be as low as 0.44 MPa for the US PNP and 4.01 mJ/cm2 for the laser fluence, which are lower than those with our previous approach (0.53 MPa and 4.95 mJ/cm2 , respectively). CONCLUSION: We demonstrated the feasibility of vaporization-based sonoporation at a lower optical and acoustic energy. It is an advantageous method that can enhance drug delivery efficiency, therapeutic safety and potentially deliver an upgraded gene therapy strategy for improved theragnosis.

Laser-scribed graphene nanofiber decorated with oil palm lignin capped silver nanoparticles: a green biosensor.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), requires a high level of attention and is one of the most infectious diseases in the air. Present methods of diagnosing TB remain ineffective owing to their low sensitivity and time consumption. In this study, we produced a green graphene nanofiber laser biosensor (LSG-NF) decorated with oil palm lignin-based synthetic silver nanoparticles (AgNPs). The resulting composite morphology was observed by field-emission scanning electron microscopy and transmission electron microscopy, which revealed the effective adaptation of the AgNPs to the LSG-NF surface. The successful attachment of AgNPs and LSG-NFs was also evident from X-ray diffraction and Raman spectroscopy studies. In order to verify the sensing efficiency, a selective DNA sample captured on AgNPs was investigated for specific binding with M.tb target DNA through selective hybridisation and mismatch analysis. Electrochemical impedance studies further confirmed sensitive detection of up to 1 fM, where a detection limit of 10-15 M was obtained by estimating the signal-to-noise ratio (S/N = 3:1) as 3σ. Successful DNA immobilisation and hybridisation was confirmed by the detection of phosphorus and nitrogen peaks based on X-ray photoelectron spectroscopy and Fourier-transform infrared spectroscopy. The stability and repeatability of the analysis were high. This approach provides an affordable potential sensing system for the determination of M. tuberculosis biomarker and thus provides a new direction in medical diagnosis.

Functional photoacoustic calcium imaging using chlorophosphonazo III in a 3D tumor cell culture.

This study demonstrates that chlorophosphonazo III (CPZ III) can be used as a contrast agent for photoacoustic calcium imaging. CPZ III can pass across the plasma membrane for labeling intracellular Ca2+ without cytotoxicity. In optical-resolution photoacoustic microscopy (OR-PAM), the photoacoustic (PA) signal intensity was strongly correlated with the presence of CPZ III and Ca2+ at various concentrations. The sensitivity of PA signal reception was enhanced by using an 8 MHz single-element focused ultrasound detector due to their matched frequency characteristics. Differences in the PA signal intensity were successfully found between the core and margin areas of tumorspheres in three-dimensional cell cultures. These findings indicate that CPZ III can serve as a novel PA contrast agent for functional Ca2+ imaging using OR-PAM.

Enhanced Radiosensitization for Cancer Treatment with Gold Nanoparticles through Sonoporation.

We demonstrate the megavoltage (MV) radiosensitization of a human liver cancer line by combining gold-nanoparticle-encapsulated microbubbles (AuMBs) with ultrasound. Microbubbles-mediated sonoporation was administered for 5 min, at 2 h prior to applying radiotherapy. The intracellular concentration of gold nanoparticles (AuNPs) increased with the inertial cavitation of AuMBs in a dose-dependent manner. A higher inertial cavitation dose was also associated with more DNA damage, higher levels of apoptosis markers, and inferior cell surviving fractions after MV X-ray irradiation. The dose-modifying ratio in a clonogenic assay was 1.56 ± 0.45 for a 10% surviving fraction. In a xenograft mouse model, combining vascular endothelial growth factor receptor 2 (VEGFR2)-targeted AuMBs with sonoporation significantly delayed tumor regrowth. A strategy involving the spatially and temporally controlled release of AuNPs followed by clinically utilized MV irradiation shows promising results that make it worthy of further translational investigations.

Photoacoustic imaging of cells in a three-dimensional microenvironment.

Imaging live cells in a three-dimensional (3D) culture system yields more accurate information and spatial visualization of the interplay of cells and the surrounding matrix components compared to using a two-dimensional (2D) cell culture system. However, the thickness of 3D cultures results in a high degree of scattering that makes it difficult for the light to penetrate deeply to allow clear optical imaging. Photoacoustic (PA) imaging is a powerful imaging modality that relies on a PA effect generated when light is absorbed by exogenous contrast agents or endogenous molecules in a medium. It combines a high optical contrast with a high acoustic spatiotemporal resolution, allowing the noninvasive visualization of 3D cellular scaffolds at considerable depths with a high resolution and no image distortion. Moreover, advances in targeted contrast agents have also made PA imaging capable of molecular and cellular characterization for use in preclinical personalized diagnostics or PA imaging-guided therapeutics. Here we review the applications and challenges of PA imaging in a 3D cellular microenvironment. Potential future developments of PA imaging in preclinical applications are also discussed.

Facile, Rapid, and Low-Cost Electrophoresis Titration of Thrombin by Aptamer-Linked Magnetic Nanoparticles and a Redox Boundary Chip.

An aptamer-linked assay of a target biomarker (e.g., thrombin) is facing the challenges of long-term run, complex performance, and expensive instrument, unfitting clinical diagnosis in resource-limited areas. Herein, a facile chip electrophoresis titration (ET) model was proposed for rapid, portable, and low-cost assay of thrombin via aptamer-linked magnetic nanoparticles (MNPs), redox boundary (RB), and horseradish peroxidase (HRP). In the electrophoresis titration-redox boundary (ET-RB) model, thrombin was chosen as a model biomarker, which could be captured within 15 min by MNP-aptamer 1 and HRP-aptamer 2, forming a sandwich complex of (MNP-aptamer 1)-thrombin-(HRP-aptamer 2). After MNP separation and chromogenic reaction of 3,3',5,5'-tetramethylbenzidine (TMB) within 10 min, an ET-RB run could be completed within 5 min based on the reaction between a 3,3',5,5'-tetramethylbenzidine radical cation (TMB•+) and l-ascorbic acid in the ET channel. The systemic experiments based on the ET-RB method revealed that the sandwich complex could be formed and the thrombin content could be assayed via an ET-RB chip, demonstrating the developed model and method. In particular, the ET-RB method had the evident merits of simplicity, rapidity (less than 30 min), and low cost as well as portability and visuality, in contrast to the currently used thrombin assay. In addition, the developed method had high selectivity, sensitivity (limit of detection of 0.04 nM), and stability (intraday: 3.26%, interday: 6.07%) as well as good recovery (urine: 97-102%, serum: 94-103%). The developed model and method have potential to the development of a point-of-care testing assay in resource-constrained conditions.

Synchronized Optical and Acoustic Droplet Vaporization for Effective Sonoporation.

Inertial cavitation-based sonoporation has been utilized to enhance treatment delivery efficacy. In our previous study, we demonstrated that tumor therapeutic efficacy can be enhanced through vaporization-assisted sonoporation with gold nanodroplets (AuNDs). Specifically, the AuNDs were vaporized both acoustically (i.e., acoustic droplet vaporization, ADV) and optically (i.e., optical droplet vaporization, ODV). A continuous wave (CW) laser was used for ODV in combination with an ultrasound pulse for ADV. Although effective for vaporization, the use of a CW laser is not energy efficient and may create unwanted heating and concomitant tissue damage. In this study, we propose the use of a pulsed wave (PW) laser to replace the CW laser. In addition, the PW laser was applied at the rarefaction phase of the ultrasound pulse so that the synergistic effects of ADV and ODV can be expected. Therefore, a significantly lower laser average power can be expected to achieve the vaporization threshold. Compared to the CW laser power at 2 W/cm2 from the previous approach, the PW laser power was reduced to only 0.2404 W/cm2. Furthermore, we also demonstrate in vitro that the sonoporation rate was increased when the PW laser was applied at the rarefaction phase. Specifically, the vaporization signal, the inertial cavitation signal, and the sonoporation rate all displayed a 1-µs period, which corresponded to the period of the 1-MHz acoustic wave used for ADV, as a function of the relative laser delay. The increased sonoporation rate indicates that this technique has the potential to enhance sonoporation-directed drug delivery and tumor therapy with a lower laser power while keeping the cell death rate at the minimum. Photoacoustic imaging can also be performed at the same time since a PW laser is used for the ODV.

Study of diffusive- and convective-transport mediated microtumor growth in a controlled microchamber.

In this paper, we report on using mass transport to control nutrition supply of colorectal cancer cells for developing a microtumor in a confined microchamber. To mimic the spatial heterogeneity of a tumor, two microfluidic configurations based on resistive circuits are designed. One has a convection-dominated microchamber to simulate the tumor region proximal to leaky blood vessels. The other has a diffusion-dominated microchamber to mimic the tumor core that lacks blood vessels and nutrient supply. Thus, the time for nutrition to fill the microchamber can vary from tens of minutes to several hours. Results show that cells cultured under a diffusive supply of nutrition have a high glycolytic rate and a nearly constant oxygen consumption rate. In contrast, cells cultured under convective supply of nutrition have a gradual increase of oxygen consumption rate with a low glycolytic rate. This suggests that cancer cells have distinct reactions under different mass transport and nutrition supply. Using these two microfluidic platforms to create different rate of nutrition supply, it is found that a continuous microtumor that almost fills the mm-size microchamber can be developed under a low-nutrient supply environment, but not for the convective condition. It also is demonstrated that microchannels can simulate the delivery of anti-cancer drugs to the microtumor under controlled mass-transport. This method provides a means to develop a larger scale microtumor in a lab-on-a-Chip system for post development and stimulations, and microchannels can be applied to control the physical and chemical environment for anti-cancer drug screening.

Electrophoresis Titration Model of a Moving Redox Boundary Chip for a Point-of-Care Test of an Enzyme-Linked Immunosorbent Assay.

Enzyme-linked immunosorbent assays (ELISAs) have been widely used in clinical examination, food safety, and environmental analyses. However, they still face a great challenge in designing a device for a point-of-care test (POCT) due to its bulk optical detector and complexity. Herein an electrophoresis titration (ET) model of a moving redox boundary (MRB) was proposed for constructing an ET-ELISA chip of a POCT just with sextuplet electrode pairs and laminated cells. The chip had an anodic well, middle well, and cathode well which were connected by microchannels. The ELISA process was conducted in the bottom of the middle well, where horseradish peroxidase (HRP) catalyzed 3,3',5,5'-tetra-methyl benzidine (TMB) as a blue TMB dimer with two positive charges. Under an electrical field of 29 V, the TMB dimer migrated into the titration channel and reacted with the ascorbic acid, creating an MRB. The MRB motion was a function of antigen content, indicating a visual distance-based assay. As a proof of concept, a C-reactive protein was chosen as a model antigen. The experiments systemically validated the ET-ELISA model and method. Particularly, the chip was smartphone-detected, traditional power supply free, and did not use sulfuric acid used in typical ELISA, making the ET-ELISA method extremely simple, portable, and safe. The ET-ELISA has great potential to visual and portable ELISA in clinical medicine, the environment, and food safety immunoassay.

Optimization and characterization of magnetite-reduced graphene oxide nanocomposites for demulsification of crude oil in water emulsion.

Oily wastewater from the oil and gas industry negatively affects the environment. Oily wastewater typically exists in the form of an oil-in-water emulsion. Conventional methods to treat oily wastewater have low separation efficiency and long separation time and use large equipment. Therefore, a simple but effective method must be developed to separate oil-in-water emulsions with high separation efficiency and short separation times. Magnetite-reduced graphene oxide (M-RGO) nanocomposites were used as a demulsifier in this work. Magnetite nanoparticles (Fe3O4) were coated on reduced graphene oxide (rGO) nanosheets via an in situ chemical synthesis method. The synthesized M-RGO nanocomposites are environmentally friendly and can be recovered after demulsification by an external magnetic field. M-RGO characterization was performed using X-ray diffraction, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, field emission scanning microscopy, Raman spectroscopy, and vibrating sample magnetometry. Demulsification performance was evaluated in terms of M-RGO dosage, effects of pH, and brine concentration. The demulsification capability of M-RGO was determined based on the residual oil content of the emulsion, which was measured with a UV-vis spectrometer. The response surface method was used to determine the optimum conditions of the input variables. The optimum demulsification efficiency achieved at pH 4 and M-RGO dosage of 29 g L-1 was approximately 96%. This finding demonstrates that M-RGO nanocomposites are potential magnetic demulsifiers for oily wastewater that contains oil-in-water emulsions. Also, the recyclability of this nanocomposite has been tested and the results shown that it is a good recyclable demulsifier.

Shear Wave Elasticity Measurements of Three-Dimensional Cancer Cell Cultures Using Laser Speckle Contrast Imaging.

Shear wave elastography (SWE) has been widely adopted for clinical in vivo imaging of tissue elasticity for disease diagnosis, and this modality can be a valuable tool for in vitro mechanobiology studies but its full potential has yet to be explored. Here we present a laser speckle contrast SWE system for noncontact monitoring the spatiotemporal changes of the extracellular matrix (ECM) stiffness in three-dimensional cancer cell culture system while providing submillimeter spatial resolution and temporal resolution of 10 s. The shear modulus measured was found to be strongly correlated with the ECM fiber density in two types of cell culture system (r = 0.832 with P < 0.001, and r = 0.642 with P = 0.024 for cell culture systems containing 4 mg/ml Matrigel with 1 mg/ml and 2 mg/ml collagen type I hydrogel, respectively). Cell migration along the stiffness gradient in the cell culture system and an association between cell proliferation and the local ECM stiffness was observed. As the elasticity measurement is performed without the need of exogenous probes, the proposed method can be used to study how the microenvironmental stiffness interacts with cancer cell behaviors without possible adverse effects of the exogenous particles, and could potentially be an effective screening tool when developing new treatment strategies.

Regulation of axon repulsion by MAX-1 SUMOylation and AP-3.

During neural development, growing axons express specific surface receptors in response to various environmental guidance cues. These axon guidance receptors are regulated through intracellular trafficking and degradation to enable navigating axons to reach their targets. In Caenorhabditis elegans, the UNC-5 receptor is necessary for dorsal migration of developing motor axons. We previously found that MAX-1 is required for UNC-5-mediated axon repulsion, but its mechanism of action remained unclear. Here, we demonstrate that UNC-5-mediated axon repulsion in C. elegans motor axons requires both max-1 SUMOylation and the AP-3 complex ß subunit gene, apb-3 Genetic interaction studies show that max-1 is SUMOylated by gei-17/PIAS1 and acts upstream of apb-3 Biochemical analysis suggests that constitutive interaction of MAX-1 and UNC-5 receptor is weakened by MAX-1 SUMOylation and by the presence of APB-3, a competitive interactor with UNC-5. Overexpression of APB-3 reroutes the trafficking of UNC-5 receptor into the lysosome for protein degradation. In vivo fluorescence recovery after photobleaching experiments shows that MAX-1 SUMOylation and APB-3 are required for proper trafficking of UNC-5 receptor in the axon. Our results demonstrate that SUMOylation of MAX-1 plays an important role in regulating AP-3-mediated trafficking and degradation of UNC-5 receptors during axon guidance.

iPhone-imaged and cell-powered electrophoresis titration chip for the alkaline phosphatase assay in serum by the moving reaction boundary.

As a vital enzyme, alkaline phosphatase (ALP) has great clinical significance in diagnoses of bone or liver cancer, bone metastases, rickets, and extrahepatic biliary obstruction. However, there is still no really portable chip for the ALP assay in blood. Herein, a simple electrophoresis titration (ET) model was developed for ALP detection via a moving reaction boundary (MRB). In the model, ALP catalyzed the dephosphorylation of a 4-methylumbelliferyl phosphate disodium salt (4-MUP) substrate in the cathode well to 4-methylumbelliferone ([4-MU]-) with a negative charge and blue fluorescence under UV excitation. After the catalysis, an electric field was used between the cathode and the anode. Under the electric field, [4-MU]- moved into the channel and neutralized the acidic Tris-HCl buffer, resulting in the quenching of [4-MU]- and creating a MRB. The ET system just had an ET chip, a lithium cell, a UV LED and an iPhone used as a recorder, having no traditional expensive power supply and fluorescence detector. The relevant method was developed, and a series of experiments were conducted via the ET chip. The experiments showed: (i) a MRB could be formed between the [4-MU]- base and the acidic buffer, and the MRB motion had a linear relationship with the ALP activity, validating the ET model; (ii) the ET run was not impacted by many interferences, implying good selectivity; and (iii) the ET chip could be used for portable detection within 10 min, implying an on-site and rapid analysis. In addition, the ET method had a relatively good sensitivity (0.1 U L-1), linearity (V = 0.033A + 3.87, R2 = 0.9980), stability (RSD 2.4-6.8%) and recoveries (101-105%). Finally, the ET method was successfully used for ALP assays in real serum samples. All the results implied that the developed method was simple, rapid and low-cost, and had potential for POCT clinical ALP assays.