Real-time imaging of photosynthetic oxygen evolution from spinach using LSI-based biosensor.

The light-driven splitting of water to oxygen (O2) is catalyzed by a protein-bound tetra-manganese penta-oxygen calcium (Mn4O5Ca) cluster in Photosystem II. In the current study, we used a large-scale integration (LSI)-based amperometric sensor array system, designated Bio-LSI, to perform two-dimensional imaging of light-induced O2 evolution from spinach leaves. The employed Bio-LSI chip consists of 400 sensor electrodes with a pitch of 250 µm for fast electrochemical imaging. Spinach leaves were illuminated to varying intensities of white light (400-700 nm) which induced oxygen evolution and subsequent electrochemical images were collected using the Bio-LSI chip. Bio-LSI images clearly showed the dose-dependent effects of the light-induced oxygen release from spinach leaves which was then significantly suppressed in the presence of urea-type herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Our results clearly suggest that light-induced oxygen evolution can be monitored using the chip and suggesting that the Bio-LSI is a promising tool for real-time imaging. To the best of our knowledge, this report is the first to describe electrochemical imaging of light-induced O2 evolution using LSI-based amperometric sensors in plants.

Electrochemical Imaging of Cell Activity in Hydrogels Embedded in Grid-shaped Polycaprolactone Scaffolds Using a Large-scale Integration-based Amperometric Device.

Tissue engineering requires analytical methods to monitor cell activity in hydrogels. Here, we present a method for the electrochemical imaging of cell activity in hydrogels embedded in printed polycaprolactone (PCL) scaffolds. Because a structure made of only hydrogel is fragile, PCL frameworks are used as a support material. A grid-shaped PCL was fabricated using an excluder printer. Photocured hydrogels containing cells were set at each grid hole, and cell activity was monitored using a large-scale integration-based amperometric device. The electrochemical device contains 400 microelectrodes for biomolecule detection, such as dissolved oxygen and enzymatic products. As proof of the concept, alkaline phosphatase and respiration activities of embryonic stem cells in the hydrogels were electrochemically monitored. The results indicate that the electrochemical imaging is useful for evaluating cells in printed scaffolds.

Electrochemicolor Imaging Using an LSI-Based Device for Multiplexed Cell Assays.

Multiplexed bioimaging systems have triggered the development of effective assays, contributing new biological information. Although electrochemical imaging is beneficial for quantitative analysis in real time, monitoring multiple cell functions is difficult. We have developed a novel electrochemical imaging system, herein, using a large-scale integration (LSI)-based amperometric device for detecting multiple biomolecules simultaneously. This system is designated as an electrochemicolor imaging system in which the current signals from two different types of biomolecules are depicted as a multicolor electrochemical image. The mode-selectable function of the 400-electrode device enables the imaging system and two different potentials can be independently applied to the selected electrodes. The imaging system is successfully applied for detecting multiple cell functions of the embryonic stem (ES) cell and the rat pheochromocytoma (PC12) cell aggregates. To the best of our knowledge, this is the first time that a real-time electrochemical mapping technique for multiple electroactive species, simultaneously, has been reported. The imaging system is a promising bioanalytical method for exploring complex biological phenomena.

Electrochemical Motion Tracking of Microorganisms Using a Large-Scale-Integration-Based Amperometric Device.

Motion tracking of microorganisms is useful to investigate the effects of chemical or physical stimulation on their biological functions. Herein, we describe a novel electrochemical imaging method for motion tracking of microorganisms using a large-scale integration (LSI)-based amperometric device. The device consists of 400 electrochemical sensors with a pitch of 250 µm. A convection flow caused by the motion of microorganisms supplies redox species to the sensors and increases their electrochemical responses. Thus, the flow is converted to electrochemical signals, enabling the electrochemical motion tracking of the microorganisms. As a proof of concept, capillary vibration was monitored. Finally, the method was applied to monitoring the motion of Daphnia magna. The motions of these microorganisms were clearly tracked based on the electrochemical oxidation of [Fe(CN)6 ]4- and reduction of O2 .

Imaging of enzyme activity using bio-LSI system enables simultaneous immunosensing of different analytes in multiple specimens.

Electrochemical imaging is an excellent technique to characterize an activity of biomaterials, such as enzymes and cells. Large scale integration-based amperometric sensor (Bio-LSI) has been developed for the simultaneous and continuous detection of the concentration distribution of redox species generated by reactions of biomolecules. In this study, the Bio-LSI system was demonstrated to be applicable for simultaneous detection of different anaytes in multiple specimens. The multiple specimens containing human immunoglobulin G (hIgG) and mouse IgG (mIgG) were introduced into each channel of the upper substrate across the antibody lines for hIgG and mIgG on the lower substrate. Hydrogen peroxide generated by the enzyme reaction of glucose oxidase captured at intersections was simultaneously detected by 400 microelectrodes of Bio-LSI chip. The oxidation current increased with increasing the concentrations of hIgG, which can be detected in the range of 0.01-1.0 µg mL(-1) . Simultaneous detection of hIgG and mIgG in multiple specimens was achieved by using line pattern of both antibodies. Therefore, the presence of different target molecules in the multiple samples would be quantitatively and simultaneously visualized as a current image by the Bio-LSI system.

Potentiometric bioimaging with a large-scale integration (LSI)-based electrochemical device for detection of enzyme activity.

This paper describes potentiometric bioimaging for enzyme activity using a large-scale integration (LSI)-based electrochemical device with 400 sensors. Potentiometric detection is useful for bioimaging because redox species are not consumed or produced during the detection process; therefore, there is no effect on cell activity and the detectable signal is sustained. In this study, the potentiometer mode of the LSI-based device was applied for the detection of glucose oxidase (GOx) and alkaline phosphatase (ALP) activity. The enzyme activities were quantitatively detected within the concentration ranges of 25-250 µg/mL and 0.10-5.0 ng/mL. In addition, GOx activity in hydrogels and the ALP activity of embryoid bodies (EBs) from embryonic stem (ES) cells were successfully imaged based on detection of the open circuit potentials of individual sensors in real time. To the best of our knowledge, this is the first report of potentiometric imaging using LSI-based electrochemical arrays to detect enzyme activity in ES cells. The LSI-based device is thus demonstrated to be a promising tool for bioimaging of enzyme activity.

Simulation Analysis of Positional Relationship between Embryoid Bodies and Sensors on an LSI-based Amperometric Device for Electrochemical Imaging of Alkaline Phosphatase Activity.

In the present study, we monitored the alkaline phosphatase (ALP) activity of embryoid bodies (EBs) of mouse embryonic stem (ES) cells using a large-scale integration (LSI)-based amperometric device with 400 sensors and a pitch of 250 µm. In addition, a simulation analysis was performed to reveal the positional relationship between the EBs and the sensor electrodes toward more precise measurements. The study shows that simulation analysis can be applied for precise electrochemical imaging of three-dimensionally cultured cells by normalization of the current signals.

Electrochemical Imaging of Dopamine Release from Three-Dimensional-Cultured PC12 Cells Using Large-Scale Integration-Based Amperometric Sensors.

In the present study, we used a large-scale integration (LSI)-based amperometric sensor array system, designated Bio-LSI, to image dopamine release from three-dimensional (3D)-cultured PC12 cells (PC12 spheroids). The Bio-LSI device consists of 400 sensor electrodes with a pitch of 250 µm for rapid electrochemical imaging of large areas. PC12 spheroids were stimulated with K(+) to release dopamine. Poststimulation dopamine release from the PC12 spheroids was electrochemically imaged using the Bio-LSI device. Bio-LSI clearly showed the effects of the dopaminergic drugs l-3,4-dihydroxyphenylalanine (L-DOPA) and reserpine on K(+)-stimulated dopamine release from PC12 spheroids. Our results demonstrate that dopamine release from PC12 spheroids can be monitored using the device, suggesting that the Bio-LSI is a promising tool for use in evaluating 3D-cultured dopaminergic cells and the effects of dopaminergic drugs. To the best of our knowledge, this report is the first to describe electrochemical imaging of dopamine release by PC12 spheroids using LSI-based amperometric sensors.

Advanced LSI-based amperometric sensor array with light-shielding structure for effective removal of photocurrent and mode selectable function for individual operation of 400 electrodes.

We have developed a large-scale integrated (LSI) complementary metal-oxide semiconductor (CMOS)-based amperometric sensor array system called "Bio-LSI" as a platform for electrochemical bio-imaging and multi-point biosensing with 400 measurement points. In this study, we newly developed a Bio-LSI chip with a light-shield structure and a mode-selectable function with the aim of extending the application range of Bio-LSI. The light shield created by the top metal layer of the LSI chip significantly reduces the noise generated by the photocurrent, whose value is less than 1% of the previous Bio-LSI without the light shield. The mode-selectable function enables the individual operation of 400 electrodes in off, electrometer, V1, and V2 mode. The off-mode cuts the electrode from the electric circuit. The electrometer-mode reads out the electrode potential. The V1-mode and the V2-mode set the selected sensor electrode at two different independent voltages and read out the current. We demonstrated the usefulness of the mode-selectable function. First, we displayed a dot picture based on the redox reactions of 2.0 mM ferrocenemethanol at 400 electrodes by applying two different independent voltages using the V1 and V2 modes. Second, we carried out a simultaneous detection of O2 and H2O2 using the V1 and V2 modes. Third, we used the off and V1 modes for the modification of the osmium-polyvinylpyridine gel polymer containing horseradish peroxidase (Os-HRP) at the selected electrodes, which act as sensors for H2O2. These results confirm that the advanced version of Bio-LSI is a promising tool that can be applied to a wide range of analytical fields.

LSI-based amperometric sensor for real-time monitoring of embryoid bodies.

A large scale integration (LSI)-based amperometric sensor is used for electrochemical evaluation and real-time monitoring of the alkaline phosphatase (ALP) activity of mouse embryoid bodies (EBs). EBs were prepared by the hanging drop culture of embryonic stem (ES) cells. The ALP activity of EBs with various sizes was electrochemically detected at 400 measurement points on a Bio-LSI chip. The electrochemical measurements revealed that the relative ALP activity was low for large EBs and decreased with progress of the differentiation level of the ES cells. The ALP activity of the EBs was successfully monitored in real time for 3.5h, and their ALP activity in a glucose-free buffer decreased after 2h. To the best of our knowledge, this is the first report on the application of an LSI-based amperometric sensor for real-time cell monitoring over 3h. The chip is expected to be useful for the evaluation of cell activities.

LSI-based amperometric sensor for bio-imaging and multi-point biosensing.

We have developed an LSI-based amperometric sensor called "Bio-LSI" with 400 measurement points as a platform for electrochemical bio-imaging and multi-point biosensing. The system is comprised of a 10.4 mm × 10.4 mm CMOS sensor chip with 20 × 20 unit cells, an external circuit box, a control unit for data acquisition, and a DC power box. Each unit cell of the chip contains an operational amplifier with a switched-capacitor type I-V converter for in-pixel signal amplification. We successfully realized a wide dynamic range from ±1 pA to ±100 nA with a well-organized circuit design and operating software. In particular, in-pixel signal amplification and an original program to control the signal read-out contribute to the lower detection limit and wide detection range of Bio-LSI. The spacial resolution is 250 µm and the temporal resolution is 18-125 ms/400 points, which depends on the desired current detection range. The coefficient of variance of the current for 400 points is within 5%. We also demonstrated the real-time imaging of a biological molecule using Bio-LSI. The LSI coated with an Os-HRP film was successfully applied to the monitoring of the changes of hydrogen peroxide concentration in a flow. The Os-HRP-coated LSI was spotted with glucose oxidase and used for bioelectrochemical imaging of the glucose oxidase (GOx)-catalyzed oxidation of glucose. Bio-LSI is a promising platform for a wide range of analytical fields, including diagnostics, environmental measurements and basic biochemistry.