One-step construction of multiplexed enzymatic biosensors using light-addressable electrochemistry on a single silicon photoelectrode.

The multiplexed detection of metabolites in parallel within a single biosensor plate is sufficiently valuable but also challenging. Herein, we combine the inherent light addressability of silicon with the high selectivity of enzymes, for the construction of multiplexed photoelectrochemical enzymatic biosensors. To conduct a stable electrochemistry and reagentless biosensing on silicon, a new strategy involving the immobilization of both redox mediators and enzymes using an amide bond-based hydrogel membrane was proposed. The membrane characterization results demonstrated a covalent coupling of ferrocene mediator to hydrogel, in which the mediator acted as not only a signal generator but also a renewable sacrifice agent. By adding corresponding enzymes on different spots of hydrogel membrane modified silicon and recording local photocurrents with a moveable light pointer, this biosensor setup was used successfully to detect multiple metabolites, such as lactate, glucose, and sarcosine, with good analytical performances. The limits of detection of glucose, sarcosine and lactate were found to be 179 µM, 16 µM, and 780 µM with the linear ranges of 0.5-2.5 mM, 0.3-1.5 mM, and 1.0-3.0 mM, respectively. We believe this proof-of-concept study provides a simple and rapid one-step immobilization approach for the fabrication of reagentless enzymatic assays with silicon-based light-addressable electrochemistry.

Label-Free Imaging of Cell Apoptosis by a Light-Addressable Electrochemical Sensor.

Label-free electrochemical visualization of cancer cell apoptosis is essential for cancer therapies. In this work, we proposed a noninvasive imaging method using a light-addressable electrochemical sensor (LAES) for label-free imaging of drug-induced tumor cell apoptosis. The dynamic AC photocurrent changes on MCF-7 human breast adenocarcinoma cells after inducing by tamoxifen were imaged. And the reasons for photocurrent changes on the cells were explored by monitoring the changes in the ζ potentials of cells and Faradic impedance. The results demonstrated that the AC photocurrent on apoptotic MCF-7 cells increased, and the apoptosis degree of each cell was heterogeneous. Moreover, the AC photocurrent increase was attributed to the increased cell membrane permeability and the increased gap between the cell basal surface and the substrate caused by cell apoptosis. This study provides a brand new approach for label-free visualizing cell apoptosis heterogeneity, which has great potential in apoptosis-associated drug screening or drug efficacy evaluation.

Carbonized polydopamine layer-protected silicon substrates for light-addressable electrochemical sensing and imaging.

The application of silicon (Si) substrate as photoelectrode in light-addressable electrochemistry (LAE) is severely limited due to its ease of surface oxidation. The resulted silicon oxide (SiOx) layer is electronically insulating and blocks charge transfer between the electrode and electrolyte. Keeping the Si from being oxidized is a key challenge for its practical use as a semiconductor electrode. In this work, we find that by developing a thin layer of polydopamine film on the surface of Si substrate, followed by carbonization at 550 °C, the natural oxidation of Si substrate can be successfully forestalled. When applied as an electrode, it is further found that the carbonized polydopamine (cPDA) layer can also prevent anodic oxidation of Si. The cPDA layer-modified Si substrate exhibits good photoelectrochemical performance and great stability, with no obvious signal decrease under ambient environment over 32 h. Our work here provides a new modification strategy for anti-oxidation of Si substrate and it is promising in the application of light-addressable electrochemical sensing and imaging.

Visualization of electrochemical reactions on microelectrodes using light-addressable potentiometric sensor imaging.

Visualization of the electrochemical reaction is essential for comprehensively understanding the electrochemical reaction mechanism and precisely characterizing dynamic electrochemical processes. Herein, we propose a simple device that combines light-addressable potentiometric sensor (LAPS) imaging and microelectrodes to serve as a general electroanalysis platform for the label-free sensing and imaging of electrochemical reactions. In this device, two microelectrodes are assembled on the LAPS chip. Electrochemical reactions occurring on the microelectrodes can be qualitatively and quantitatively observed and visualized using a LAPS chip that is sensitive to the reaction products. Validations were performed to monitor the effect of water electrolysis and potassium ferrocyanide oxidation surrounding the microelectrodes, respectively. We believe that this study will provide an excellent platform for the visualization and monitoring of electrochemical reactions and broaden the application scope of LAPS imaging to a general electroanalysis tool that is widely applicable in several fields.

Light-Addressable Electrochemical Sensors toward Spatially Resolved Biosensing and Imaging Applications.

The light-addressable electrochemical sensor (LAES) is a recently emerged bioanalysis technique combining electrochemistry with the photoelectric effect in a semiconductor. In an LAES, a semiconductor substrate is illuminated locally to generate charge carriers in a well-defined area, thereby confining the electrochemical process to a target site. Benefiting from the unique light addressability, an LAES can not only detect multiple analytes in parallel within a single sensor plate but also act as a bio(chemical) imaging sensor to visualize the two-dimensional distribution of specific analytes. An LAES usually has three working modes: a potentiometric mode using light-addressable potentiometric sensors (LAPS) and an impedance mode using scanning photoinduced impedance microscopy (SPIM), while an amperometric mode refers to light-addressable electrochemistry (LAE) and photoelectrochemical (PEC) sensing. In this review, we describe the detection principles of each mode of LAESs and the concept of light addressability. In addition, we highlight the recent progress and advance of LAESs in spatial resolution, sensor system design, multiplexed detection, and bio(chemical) imaging applications. An outlook on current research challenges and future prospects is also presented.

A Homogeneous Label-Free Electrochemical microRNA Biosensor Coupling With G-Triplex/Methylene Blue Complex and λ-Exonuclease-Assisted Recycling Amplification.

A novel homogeneous label-free electrochemical biosensor using G-triplex/methylene blue (G3/MB) complex as the signal generator together with an amplification assisted by the λ-exonuclease (λ-Exo) has been successfully constructed for ultrasensitive microRNA (miRNA) detection. An integrated microelectrode was designed to realize the miniaturization of the homogeneous electrochemical assay. Taking advantage of G3, that can specifically bind with MB and decrease its diffusion current, a single-stranded functional DNA hairpin structure was designed as the bio-recognition probe. The probe consisted of G3, eight bases to block G3, and the complementary sequences of the target miRNA. Here we chose miRNA141-a potentially diagnostic biomarker of prostate cancer as the model target. The presence of miRNA141 could hybridize with the probe DNA to form a double-stranded structure with a 5'-phosphorylated terminus. Then λ-Exo was adopted to digest mononucleotides from the 5'-end, leading to the release of G3 part and miRNA141. The released miRNA could hybridize with another probe to trigger the cycling process, while the released G3 could therefore interact with MB to cause a detectable decrease of diffusion current. The proposed strategy showed a low detection limit of 16 fM and an excellent specificity to discriminate single-base mismatches. Furthermore, this sensor was applied to detect miRNA141 from diluted human serum samples, indicating that it has great potential in the application of nucleic acid detection in real samples.

A novel miniaturized homogeneous label-free electrochemical biosensing platform combining integrated microelectrode and functional nucleic acids.

A miniaturized platform combining integrated microelectrode (IME) and functional nucleic acids was developed for homogeneous label-free electrochemical biosensing. IME was constructed with a carbon fiber microelectrode and a platinum wire in a θ type glass tube as a two-electrode system for electrochemical monitoring at microliter level. A newly reported G-triplex/methylene blue (G3/MB) complex was used as the signal generator in the homogeneous label-free electrochemical biosensor. G3 has strong affinity with MB and it can cause significant decrease of the diffusion current of MB after binding. Melamine was chosen as the model target. Since melamine can interact with nucleobase thymine (T) to form T-melamine-T structure through complementary hydrogen bonds, a single-strand functional DNA hairpin structure with poly T and G3 elaborately blocked via base pairing was designed. The presence of melamine can trigger the conformation switching of the DNA hairpin to release the G3. The released G3 combined with MB could therefore change the diffusion current, leading to a simple and rapid detection of melamine. The combination of functional DNA hairpin as target recognition element, G3/MB as signal generator, and IME as transducer provided a "Mix and Measure" miniaturized platform for the construction of homogeneous label-free electrochemical biosensors.

Light-Addressable Square Wave Voltammetry (LASWV) Based on a Field-Effect Structure for Electrochemical Sensing and Imaging.

Here, we describe a new photoelectrochemical imaging method termed light-addressable square wave voltammetry (LASWV). It measures local SWV currents at an unstructured electrolyte/insulator/semiconductor (EIS) field-effect substrate by illuminating and addressing the substrate with an intensity-constant laser. Due to the continuous generation of charge carriers in the light-irradiated semiconductor, the drift and diffusion of photoinjected carriers within the semiconductor bulk would slow down the equilibrium processes of charge and discharge in one potential pulse cycle. Therefore, even though SWV is sampled at the end of the direct and reverse pulses to reject capacitive currents, in our approach, photoinduced capacitive current can still be detected as an effective sensory signal. The obtained current-potential (I-V) curve shows a typical shape corresponding to the accumulation, depletion, and inversion regions of field-effect devices. We demonstrated that LASWV can be used as a field-effect chemical sensor to measure the solution pH and monitor enzymatic reactions. More importantly, since the charge carriers are only generated in the illuminated area, the laser spot in the device can be used as a virtual probe to record local electrochemical properties such as impedance with microresolution.

Photoelectrochemical imaging system with high spatiotemporal resolution for visualizing dynamic cellular responses.

Photoelectrochemical imaging has great potential in the label-free investigation of cellular processes. Herein, we report a new fast photoelectrochemical imaging system (PEIS) for DC photocurrent imaging of live cells, which combines high speed with excellent lateral resolution and high photocurrent stability, which are all crucial for studying dynamic cellular processes. An analog micromirror was adopted to raster the sensor substrate, enabling high-speed imaging. α-Fe2O3 (hematite) thin films synthesized via electrodeposition were used as a robust substrate with high photocurrent and good spatial resolution. The capabilities of this system were demonstrated by monitoring cell responses to permeabilization with Triton X-100. The ability to carry out dynamic functional imaging of multiple cells simultaneously provides improved confidence in the data than could be achieved with the slower electrochemical single-cell imaging techniques described previously. When monitoring pH changes, the PEIS can achieve frame rates of 8 frames per second.

Ammonia Gas Sensor Response of a Vertical Zinc Oxide Nanorod-Gold Junction Diode at Room Temperature.

Conventional metal oxide semiconductor (MOS) gas sensors have been investigated for decades to protect our life and property. However, the traditional devices can hardly fulfill the requirements of our fast developing mobile society, because the high operating temperatures greatly limit their applications in battery-loaded portable systems that can only drive devices with low power consumption. As ammonia is gaining importance in the production and storage of hydrogen, there is an increasing demand for energy-efficient ammonia detectors. Hence, in this work, a Schottky diode resulting from the contact between zinc oxide nanorods and gold is designed to detect gaseous ammonia at room temperature with a power consumption of 625 µW. The Schottky diode gas sensors benefit from the change of barrier height in different gases as well as the catalytic effect of gold nanoparticles. This diode structure, fabricated without expensive interdigitated electrodes and displaying excellent performance at room temperature, provides a novel method to equip mobile devices with MOS gas sensors.

Scanning Electrochemical Photometric Sensors for Label-Free Single-Cell Imaging and Quantitative Absorption Analysis.

A new photoelectrochemical imaging method termed scanning electrochemical photometric sensor (SEPS) is proposed in this work. It was derived from light-addressable potentiometric sensor (LAPS) and scanning photoinduced impedance microscopy (SPIM) using a front-side laser illumination at a field-effect structure. When the laser beam scans across the sensor substrate, local photocurrent changes at inversion due to the light absorption of analytes can be recorded. It will be shown that SEPS could be used for label-free living cell imaging with micro-resolution as well as real-time quantitative absorption analysis, which would broaden the applications of traditional LAPS/SPIM from potentiometric/impedance measurements to local optical analysis.

[Effect of acupuncture at " three points of ilioumbar" on lumbar function and pain in patients with iliopsoas muscle strain].

OBJECTIVE: To compare the clinical effect of acupuncture at "three points of iliolumbar" combined with celecoxib and celecoxib alone in the treatment of iliopsoas muscle strain. METHODS: A total of 60 patients with iliopsoas muscle strain were randomly divided into an observation group and a control group, 30 patients in each group. Celecoxib was given orally to both groups, 200 mg once a day for 3 days. On the basis of the above drugs, acupuncture was applied at Yaoda (Extra), Wushu (GB 27), Qiayao (Extra) in the observation group, once a day for 3 days. The Japanese Orthopaedic Association (JOA) score and visual analogue scale (VAS) score were observed and compared before and after treatment, and the content of 5-hydroxytryptamine (5-HT) in serum was detected by enzyme-linked immunosorbent assay (ELISA) before and after treatment in the two groups. RESULTS: After treatment, the JOA scores in the two groups were increased (both P<0.05), and the VAS scores and 5-HT contents were decreased (all P<0.05). The increase of JOA score in the observation group was greater than that in the control group (P<0.05), and the decrease of VAS score and 5-HT content in the observation group was greater than that in the control group (both P<0.05). CONCLUSION: Acupuncture at "three points of ilioumbar" combined with celecoxib in the treatment of iliopsoas muscle strain can improve lumbar function, relieve pain and reduce 5-HT content in serum, which is better than celecoxib alone.

Modulated light-activated electrochemistry at silicon functionalized with metal-organic frameworks towards addressable DNA chips.

Modulated light-activated electrochemistry (MLAE) at semiconductor/liquid interfaces derived from light-addressable potentiometric sensor (LAPS) and light-activated electrochemistry (LAE) for addressable photoelectrochemical sensing has been proposed as a new sensor platform. In this system, a bias voltage is applied to create a depletion layer at the silicon/electrolyte interface. Meanwhile, intensity-modulated light illuminates the movable electrode to generate electron/hole pairs and causes a detectable local AC photocurrent. The AC measurement showed a higher signal-to-noise ratio (SNR) of photocurrents compared to the traditional DC response, while a steeper photocurrent-voltage (I-V) curve than that of LAPS with an insulating layer was obtained. Furthermore, to stabilize and functionalize the silicon substrate, metal-organic framework (MOF) nanoparticles were grown in-situ on the silicon electrode. The successful modification was validated by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The AC photocurrent increased as a result of the adsorption of negatively charged DNA, which contributed to the enhancement of the cathodic reduction process at the semiconductor electrodes, indicating a different response mechanism of MLAE from LAPS. The results obtained demonstrate the potential of MOF functionalized MLAE as a robust platform for light-addressable DNA chips with high sensitivity and specificity.

A bioelectronic taste sensor based on bioengineered Escherichia coli cells combined with ITO-constructed electrochemical sensors.

In this study, we developed a novel bioelectronic taste sensor for the detection of specific bitter substances. A human bitter taste receptor, hT2R4, was efficiently expressed in Escherichia coli (E. coli), which was used as the primary recognition element. A simple and low-cost electrochemical device based on ITO-based electrolyte-semiconductor (ES) structure was innovatively employed as the transducer to assess bacterial metabolic consequences of receptor activation in real time. An apparent increase in extracellular acidification rate was observed, which was resulted from the triggering of hT2R4 receptors by their target ligand of denatonium. The sensor showed dose-dependent responses to denatonuim ranging from 50 nM to 500 nM, while non-bioengineered bacteria without hT2R4 receptors exhibited negligible responses to the same stimulus. In addition, the specificity of the proposed taste biosensor was verified using other typical bitter substances such as quinine and alpha-naphthylthiourea (ANTU). This research provides a simple and inexpensive approach for the construction of bioelectronic taste sensors.

Peptide Cross-Linked Poly(2-oxazoline) as a Sensor Material for the Detection of Proteases with a Quartz Crystal Microbalance.

Inflammatory conditions are frequently accompanied by increased levels of active proteases, and there is rising interest in methods for their detection to monitor inflammation in a point of care setting. In this work, new sensor materials for disposable single-step protease biosensors based on poly(2-oxazoline) hydrogels cross-linked with a protease-specific cleavable peptide are described. The performance of the sensor material was assessed targeting the detection of matrix metalloproteinase-9 (MMP-9), a protease that has been shown to be an indicator of inflammation in multiple sclerosis and other inflammatory conditions. Films of the hydrogel were formed on gold-coated quartz crystals using thiol-ene click chemistry, and the cross-link density was optimized. The degradation rate of the hydrogel was monitored using a quartz crystal microbalance (QCM) and showed a strong dependence on the MMP-9 concentration. A concentration range of 0-160 nM of MMP-9 was investigated, and a lower limit of detection of 10 nM MMP-9 was determined.

Photoelectrochemical Imaging System for the Mapping of Cell Surface Charges.

The surface charge of cells affects cell signaling, cell metabolic processes, adherence to surfaces, and cell proliferation. Our understanding of the role of membrane charges is limited due to the inability to observe changes without interfering, chemically or physically, with the cell or its membrane. Here, we report that a photoelectrochemical imaging system (PEIS) based on label-free ac-photocurrent measurements at indium tin oxide (ITO) coated glass substrates can be used to map the basal surface charge of single live cells under physiological conditions. Cells were cultured on the ITO substrate. Photocurrent images were generated by scanning a focused, modulated laser beam across the back of the ITO coated glass substrate under an applied bias voltage. The photocurrent was shown to be sensitive to the negative surface charge of the substrate facing, basal side of a single living cell-an area not accessible to other electrochemical or electrophysiological imaging techniques. The PEIS was used to monitor the lysis of mesenchymal stem cells.

Peptide Cross-Linked Poly (Ethylene Glycol) Hydrogel Films as Biosensor Coatings for the Detection of Collagenase.

Peptide cross-linked poly(ethylene glycol) hydrogel has been widely used for drug delivery and tissue engineering. However, the use of this material as a biosensor for the detection of collagenase has not been explored. Proteases play a key role in the pathology of diseases such as rheumatoid arthritis and osteoarthritis. The detection of this class of enzyme using the degradable hydrogel film format is promising as a point-of-care device for disease monitoring. In this study, a protease biosensor was developed based on the degradation of a peptide cross-linked poly(ethylene glycol) hydrogel film and demonstrated for the detection of collagenase. The hydrogel was deposited on gold-coated quartz crystals, and their degradation in the presence of collagenase was monitored using a quartz crystal microbalance (QCM). The biosensor was shown to respond to concentrations between 2 and 2000 nM in less than 10 min with a lower detection limit of 2 nM.

Light-Addressable Potentiometric Sensors Using ZnO Nanorods as the Sensor Substrate for Bioanalytical Applications.

Light-addressable potentiometric sensors (LAPS) are of great interest in bioimaging applications such as the monitoring of concentrations in microfluidic channels or the investigation of metabolic and signaling events in living cells. By measuring the photocurrents at electrolyte-insulator-semiconductor (EIS) and electrolyte-semiconductor structures, LAPS can produce spatiotemporal images of chemical or biological analytes, electrical potentials and impedance. However, its commercial applications are often restricted by their limited AC photocurrents and resolution of LAPS images. Herein, for the first time, the use of 1D semiconducting oxides in the form of ZnO nanorods for LAPS imaging is explored to solve this issue. A significantly increased AC photocurrent with enhanced image resolution has been achieved based on ZnO nanorods, with a photocurrent of 45.7 ± 0.1 nA at a light intensity of 0.05 mW, a lateral resolution as low as 3.0 µm as demonstrated by images of a PMMA dot on ZnO nanorods and a pH sensitivity of 53 mV/pH. The suitability of the device for bioanalysis and bioimaging was demonstrated by monitoring the degradation of a thin poly(ester amide) film with the enzyme α-chymotrypsin using LAPS. This simple and robust route to fabricate LAPS substrates with excellent performance would provide tremendous opportunities for bioimaging.

Clinical significance of imaging findings for atlantoaxial rotatory subluxation in children

Background/aim: This study evaluated the usefulness of measuring the atlantodental interval (ADI) and lateral atlantodental spaces (LADSs) by retrospectively analyzing the imaging data of children of various ages with and without atlantoaxial rotatory subluxation (AARS). Materials and methods: The data of 495 children who underwent cranial computed tomography were collected. Among these children, 255 children were clinically diagnosed with AARS while 240 children were not. ADI and LADS values were measured. Results: The difference in mean ADI between children with and without AARS in all age groups was statistically significant (t-test, all P < 0.05). In children without AARS, the 95% reference range for ADI was <3.2 mm. The difference in the variance of bilateral lateral atlantodental spaces (VBLADSs) between children with and without AARS in all age groups was statistically significant (rank-sum test, all P < 0.05), with a 95% reference range of 0­2.20 mm (D-test). Conclusion: The ADI is a well-known and frequently used parameter and is not new, but there is little research about this in China and here we confirm its application in Chinese children. The LADS is not a frequently used parameter, but it does not significantly differ. The VBLADS parameter is new and significant in this manuscript. We concluded that VBLADSs of >2.2 mm are useful indicators for the diagnosis of AARS in patients ≤14 years old. Finally, it can be concluded that ADI of ≥3.2 mm and VBLADS of >2.2 mm are useful indicators for the diagnosis of AARS in patients ≤14 years old. Widened LADSs would not be helpful in the diagnosis of AARS in children.

Interleukin-6 is a key factor for immunoglobulin-like transcript-4-mediated immune injury in sepsis.

BACKGROUND: ILT4+ monocytes seem to be associated with poor prognosis of sepsis in humans, but the exact mechanisms are unknown. This study aimed to examine the biological behaviors and effects of immunoglobulin-like transcript-4 (ILT4) levels on monocytes during sepsis and on the prognosis of sepsis. METHODS: ILT4+/+ (WT) and ILT4-knockout (ILT4-/-) male BALB/c mice were used for sepsis modeling using cecal ligation puncture (CLP). Flow cytometry was used to measure the levels of ILT4 and major histocompatibility complex class II (MHC-II) on peripheral blood monocytes 24 h after CLP. ELISA was used to measure the serum levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, IL-6, and IL-12 at 0, 6, 12, and 24 h after CLP. Survival and prognosis were monitored over the course of 168 h. RESULTS: ILT4 was highly expressed in peripheral blood monocytes of septic mice 24 h after CLP (1292.00 ± 143.70 vs. 193.50 ± 52.54, p < 0.05). MHC-II levels on peripheral blood monocytes in ILT4-/- mice were significantly higher than those in WT mice (49.38 ± 5.66% vs. 24.25 ± 6.76%, p < 0.05). Serum IL-6 was significantly elevated 24 h after CLP (470.75 ± 88.03 vs. 54.25 ± 20.04, p < 0.05). The serum IL-6 levels were significantly lower in ILT4-/- mice compared with those in WT mice after CLP (241.25 ± 45.10 vs. 470.75 ± 88.03, p < 0.05), but TNF-α, IL-1ß, and IL-12 were not changed. The survival of ILT4-/- mice was significantly better after CLP compared with that of WT mice. CONCLUSIONS: High levels of ILT4 on monocytes were observed in peripheral blood during sepsis and found to be associated with high serum IL-6 levels and low MHC-II levels on monocytes, possibly associated with higher mortality. ILT-4-IL-6-MHC-II could be a potential signaling pathway involved in sepsis.