Bio-Interface on Freestanding Nanosheet of Microelectromechanical System Optical Interferometric Immunosensor for Label-Free Attomolar Prostate Cancer Marker Detection.

Various biosensors that are based on microfabrication technology have been developed as point-of-care testing devices for disease screening. The Fabry-Pérot interferometric (FPI) surface-stress sensor was developed to improve detection sensitivity by performing label-free biomarker detection as a nanomechanical deflection of a freestanding membrane to adsorb the molecules. However, chemically functionalizing the freestanding nanosheet with excellent stress sensitivity for selective molecular detection may cause the surface chemical reaction to deteriorate the nanosheet quality. In this study, we developed a minimally invasive chemical functionalization technique to create a biosolid interface on the freestanding nanosheet of a microelectromechanical system optical interferometric surface-stress immunosensor. For receptor immobilization, glutaraldehyde cross-linking on the surface of the amino-functionalized parylene membrane reduced the shape variation of the freestanding nanosheet to 1/5-1/10 of the previous study and achieved a yield of 95%. In addition, the FPI surface-stress sensor demonstrated molecular selectivity and concentration dependence for prostate-specific antigen with a dynamic range of concentrations from 100 ag/mL to 1 µg/mL. In addition, the minimum limit of detection of the proposed sensor was 2,000,000 times lower than that of the conventional nanomechanical cantilevers.

A Multimodal Sensing Device for Simultaneous Measurement of Dissolved Oxygen and Hydrogen Ions by Monolithic Integration of FET-Based Sensors.

We examined the possibility of measuring dissolved oxygen by using a potentiometric solid-state semiconductor sensor. Thin films of tin (IV) oxide (SnO2) are widely used in oxygen gas sensors. However, their ability to detect dissolved oxygen (DO) in solutions is still unknown. In this paper, we present a method for investigating the dissolved oxygen-sensing properties of SnO2 thin films in solutions by fabricating a SnO2-gate field-effect transistor (FET). A similarly structured hydrogen ion-sensitive silicon nitride (Si3N4)-gate FET was fabricated using the same method. The transfer characteristics and sensitivities were experimentally obtained and compared. The transfer characteristics of the FET show a shift in threshold voltage in response to a decrease in DO concentration. The SnO2-gate FET exhibited a sensitivity of 4 mV/ppm, whereas the Si3N4-gate FET showed no response to DO. Although the SnO2-gate FET responds to pH changes in the solution, this sensitivity issue can be eliminated by using a Si3N4-gate FET, which is capable of selectively sensing hydrogen ions without DO sensitivity. The experimental results indicate the promising properties of SnO2 thin films for multimodal sensing applications.

CMOS-Based Redox-Type Label-Free ATP Image Sensor for In Vitro Sensitive Imaging of Extracellular ATP.

Adenosine 5'-triphosphate (ATP) plays a crucial role as an extracellular signaling molecule in the central nervous system and is closely related to various nerve diseases. Therefore, label-free imaging of extracellular ATP dynamics and spatiotemporal analysis is crucial for understanding brain function. To decrease the limit of detection (LOD) of imaging extracellular ATP, we fabricated a redox-type label-free ATP image sensor by immobilizing glycerol-kinase (GK), L-α-glycerophosphate oxidase (LGOx), and horseradish peroxidase (HRP) enzymes in a polymer film on a gold electrode-modified potentiometric sensor array with a 37.3 µm-pitch. Hydrogen peroxide (H2O2) is generated through the enzymatic reactions from GK to LGOx in the presence of ATP and glycerol, and ATP can be detected as changes in its concentration using an electron mediator. Using this approach, the LOD for ATP was 2.8 µM with a sensitivity of 77 ± 3.8 mV/dec., under 10 mM working buffers at physiological pH, such as in in vitro experiments, and the LOD was great superior 100 times than that of the hydrogen ion detection-based image sensor. This redox-type ATP image sensor may be successfully applied for in vitro sensitive imaging of extracellular ATP dynamics in brain nerve tissue or cells.

Image sensor pixel with on-chip high extinction ratio polarizer based on 65-nm standard CMOS technology.

In this study, we demonstrate a polarization sensitive pixel for a complementary metal-oxide-semiconductor (CMOS) image sensor based on 65-nm standard CMOS technology. Using such a deep-submicron CMOS technology, it is possible to design fine metal patterns smaller than the wavelengths of visible light by using a metal wire layer. We designed and fabricated a metal wire grid polarizer on a 20 × 20 µm(2) pixel for image sensor. An extinction ratio of 19.7 dB was observed at a wavelength 750 nm.

CMOS image sensor integrated with micro-LED and multielectrode arrays for the patterned photostimulation and multichannel recording of neuronal tissue.

We developed a complementary metal oxide semiconductor (CMOS) integrated device for optogenetic applications. This device can interface via neuronal tissue with three functional modalities: imaging, optical stimulation and electrical recording. The CMOS image sensor was fabricated on 0.35 µm standard CMOS process with built-in control circuits for an on-chip blue light-emitting diode (LED) array. The effective imaging area was 2.0 × 1.8 mm². The pixel array was composed of 7.5 × 7.5 µm² 3-transistor active pixel sensors (APSs). The LED array had 10 × 8 micro-LEDs measuring 192 × 225 µm². We integrated the device with a commercial multichannel recording system to make electrical recordings.

Demonstrating a Filter-Free Wavelength Sensor with Double-Well Structure and Its Application.

This study proposed a filter-free wavelength sensor with a double-well structure for detecting fluorescence without an optical filter. The impurity concentration was optimized and simulated to form a double-well-structured sensor, of which the result was consistent with the fabricated sensor. Furthermore, we proposed a novel wavelength detection method using the current ratio based on the silicon absorption coefficient. The results showed that the proposed method successfully detected single wavelengths in the 460-800 nm range. Additionally, we confirmed that quantification was possible using the current ratio of the sensor for a relatively wide band wavelength, such as fluorescence. Finally, the fluorescence that was emitted from the reagents ALEXA488, 594, and 680 was successfully identified and quantified. The proposed sensor can detect wavelengths without optical filters, which can be used in various applications in the biofield, such as POCT as a miniaturized wavelength detection sensor.

Improved Charge Pump Design and Ex Vivo Experimental Validation of CMOS 256-Pixel Photovoltaic-Powered Subretinal Prosthetic Chip.

An improved design of CMOS 256-pixel photovoltaic-powered implantable chip for subretinal prostheses is presented. In the proposed subretinal chip, a high-efficiency fully-integrated 4× charge pump is designed and integrated with on-chip photovoltaic (PV) cells and a 256-pixel array with active pixel sensors (APS) for image light sensing, biphasic constant current stimulators, and electrodes. Thus the PV voltage generated by infrared (IR) light can be boosted to above 1V so that the charge injection is increased. The proposed chip adopts the 32-phase divisional power supply scheme (DPSS) to reduce the required supply current and thus the required area of the PV cells. The proposed chip is designed and fabricated in 180-nm CMOS image sensor (CIS) technology and post-processed with biocompatible IrOx electrodes and silicone packaging. From the electrical measurement results, the measured stimulation frequency is 28.3 Hz under the equivalent electrode impedance load. The measured maximum output stimulation current is 7.1 µA and the amount of injected charges per pixel is 7.36 nC under image light intensity of 3200 lux and IR light intensity of 100 mW/cm2. The function of the proposed chip has been further validated successfully with the ex vivo experimental results by recording the electrophysiological responses of retinal ganglion cells (RGCs) of retinas from retinal degeneration (rd1) mice with a multi-electrode array (MEA). The measured average threshold injected charge is about 3.97 nC which is consistent with that obtained from the patch clamp recording on retinas from wild type (C57BL/6) mice with a single electrode pair.

Implantable CMOS Biomedical Devices.

The results of recent research on our implantable CMOS biomedical devices are reviewed. Topics include retinal prosthesis devices and deep-brain implantation devices for small animals. Fundamental device structures and characteristics as well as in vivo experiments are presented.

Chronic brain blood-flow imaging device for a behavioral experiment using mice.

A chronic brain blood-flow imaging device was developed for cerebrovascular disease treatment. This device comprises a small complementary metal-oxide semiconductor image sensor and a chronic fiber-optic plate window on a mouse head. A long-term cerebral blood-flow imaging technique was established in a freely moving mouse. Brain surface images were visible for one month using the chronic FOP window. This device obtained brain surface images and blood-flow velocity. The blood-flow changes were measured in behavioral experiments using this device. The chronic brain blood-flow imaging device may contribute to determining the cause of cerebrovascular disease and the development of cerebrovascular disease treatment.

Highly sensitive lens-free fluorescence imaging device enabled by a complementary combination of interference and absorption filters.

We report a lens-free fluorescence imaging device using a composite filter composed of an interference filter and an absorption filter, each applied to one side of a fiber optic plate (FOP). The transmission of angled excitation light through the interference filter is absorbed by the absorption filter. The auto-fluorescence of the absorption filter is reduced by the reflection from the interference filter of normally incident excitation light. As a result, high-performance rejection of excitation light is achieved in a lens-free device. The FOP provides a flat, hard imaging device surface that does not degrade the spatial resolution. We demonstrate excitation rejection of approximately 108:1 at a wavelength of 450 nm in a fabricated lens-free device. The resolution of fluorescence imaging is approximately 12 µm. Time-lapse imaging of cells containing green fluorescent protein was performed in a 5-µm thin-film chamber. The small dimensions of the device allow observation of cell culturing in a CO2 incubator. We also demonstrate that the proposed lens-free filter is compatible with super-resolution bright-field imaging techniques. These features open a way to develop a high-performance, dual-mode, lens-free imaging device that is expected to be a powerful tool for many applications, such as imaging of labeled cells and point-of-care assay.

Compact Lensless Fluorescence Counting System for Single Molecular Assay.

In digital enzyme-linked immunosorbent assay, which is used for biomarker detection and diagnosis, the concentration of target biomarkers is estimated by counting the number of fluorescence chambers in the microchamber array. We propose a compact system for counting fluorescent chambers. Our system consists of three components: a micro-reaction chamber array, an absorption filter for attenuating excitation light, and a photodetector. The absorption filter has a micro-light-pipe array (m-LPA) structure. A stacked photodiode CMOS image sensor (CIS), which can discriminate color, is applied as a photodetector. This paper describes the fabrication process enabling thin m-LPA chips. The unique low-noise characteristics of the stacked photodiode CIS that attains high sensitivity by adopting the 4T-APS configuration are explained. Furthermore, a detection method using the photobleaching phenomenon is proposed for high-sensitivity fluorescence detection. This method suggests that fluorescence by a single molecular enzyme can be detected within 30 min of the start of the fluorescence reaction.

CMOS-based opto-electronic neural interface devices for optogenetics.

CMOS-based opto-electronic neural interface devices are presented. The devices are designed with target application of in vitro and in vivo optogenetics. Two types of the opto-electronic neural interface devices are presented. One is single-chip type device for on-chip optogenetics, and the other is multi-chip type device with flexibility and wide-area coverage for in vivo optogenetics on brain. Design, packaging and functional evaluations are presented.

"Optical communication with brain cells by means of an implanted duplex micro-device with optogenetics and Ca(2+) fluoroimaging".

To better understand the brain function based on neural activity, a minimally invasive analysis technology in a freely moving animal is necessary. Such technology would provide new knowledge in neuroscience and contribute to regenerative medical techniques and prosthetics care. An application that combines optogenetics for voluntarily stimulating nerves, imaging to visualize neural activity, and a wearable micro-instrument for implantation into the brain could meet the abovementioned demand. To this end, a micro-device that can be applied to the brain less invasively and a system for controlling the device has been newly developed in this study. Since the novel implantable device has dual LEDs and a CMOS image sensor, photostimulation and fluorescence imaging can be performed simultaneously. The device enables bidirectional communication with the brain by means of light. In the present study, the device was evaluated in an in vitro experiment using a new on-chip 3D neuroculture with an extracellular matrix gel and an in vivo experiment involving regenerative medical transplantation and gene delivery to the brain by using both photosensitive channel and fluorescent Ca(2+) indicator. The device succeeded in activating cells locally by selective photostimulation, and the physiological Ca(2+) dynamics of neural cells were visualized simultaneously by fluorescence imaging.

Fabrication and functional demonstration of a smart electrode with a built-in CMOS microchip for neural stimulation of a retinal prosthesis.

In this study, we propose an advanced architecture of a smart electrode for neural stimulation of a retinal prosthesis. A feature of the proposed architecture is embedding CMOS microchips into the core of the stimulus electrodes. Microchip integration without dead space on the array is possible. Additionally, higher durability can be expected because the microchips are protected by the stimulus electrodes like a metal casing. Dedicated circular-shaped CMOS microchips were designed and fabricated. The microchip measured 400 µm in diameter. Stimulus electrodes that had a microcavity for embedding the microchip were also fabricated. In the assembly process, the CMOS microchip was mounted on a flexible substrate, and then the stimulus electrode was mounted to cover the microchip. The microchip was completely built into the inside of the electrode. By performing an ex-vivo experiment using the extracted eyeball of a pig, stimulus function of the electrode was demonstrated successfully.

Intravital fluorescence imaging of mouse brain using implantable semiconductor devices and epi-illumination of biological tissue.

The application of the fluorescence imaging method to living animals, together with the use of genetically engineered animals and synthesized photo-responsive compounds, is a powerful method for investigating brain functions. Here, we report a fluorescence imaging method for the brain surface and deep brain tissue that uses compact and mass-producible semiconductor imaging devices based on complementary metal-oxide semiconductor (CMOS) technology. An image sensor chip was designed to be inserted into brain tissue, and its size was 1500 × 450 µm. Sample illumination is also a key issue for intravital fluorescence imaging. Hence, for the uniform illumination of the imaging area, we propose a new method involving the epi-illumination of living biological tissues, and we performed investigations using optical simulations and experimental evaluation.

CMOS image sensor-based implantable glucose sensor using glucose-responsive fluorescent hydrogel.

A CMOS image sensor-based implantable glucose sensor based on an optical-sensing scheme is proposed and experimentally verified. A glucose-responsive fluorescent hydrogel is used as the mediator in the measurement scheme. The wired implantable glucose sensor was realized by integrating a CMOS image sensor, hydrogel, UV light emitting diodes, and an optical filter on a flexible polyimide substrate. Feasibility of the glucose sensor was verified by both in vitro and in vivo experiments.

Functional brain fluorescence plurimetry in rat by implantable concatenated CMOS imaging system.

Measurement of brain activity in multiple areas simultaneously by minimally invasive methods contributes to the study of neuroscience and development of brain machine interfaces. However, this requires compact wearable instruments that do not inhibit natural movements. Application of optical potentiometry with voltage-sensitive fluorescent dye using an implantable image sensor is also useful. However, the increasing number of leads required for the multiple wired sensors to measure larger domains inhibits natural behavior. For imaging broad areas by numerous sensors without excessive wiring, a web-like sensor that can wrap the brain was developed. Kaleidoscopic potentiometry is possible using the imaging system with concatenated sensors by changing the alignment of the sensors. This paper describes organization of the system, evaluation of the system by a fluorescence imaging, and finally, functional brain fluorescence plurimetry by the sensor. The recorded data in rat somatosensory cortex using the developed multiple-area imaging system compared well with electrophysiology results.

Implantable image sensor based on intra-brain image transmission.

We developed and fabricated a micro-imager based on wireless intra-brain communication using conductive property of living tissues. An pixel array, analog-to-digital converter and transmitter are integrated on a single chip. The dimensions of the chip are 1 mm × 1mm × 0.15 mm. We demonstrate wireless image transmission through phosphate buffer saline as a brain phantom.

Baseband signal transmission experiment for intra-brain communication with implantable image sensor.

We demonstrate image signal transmission for wireless intra-brain communication. As a preliminary experiment, transmission characteristics of the brain phantom were measured. The baseband output signal from an implantable complementary metal-oxide-semiconductor (CMOS) image sensor is transmitted through the phantom. The image was successfully reproduced from the received signal.

Novel implantable imaging system for enabling simultaneous multiplanar and multipoint analysis for fluorescence potentiometry in the visual cortex.

Techniques for fast, noninvasive measurement of neuronal excitability within a broad area will be of major importance for analyzing and understanding neuronal networks and animal behavior in neuroscience field. In this research, a novel implantable imaging system for fluorescence potentiometry was developed using a complementary metal-oxide semiconductor (CMOS) technology, and its application to the analysis of cultured brain slices and the brain of a living mouse is described. A CMOS image sensor, small enough to be implanted into the brain, with light-emitting diodes and an absorbing filter was developed to enable real-time fluorescence imaging. The sensor, in conjunction with a voltage-sensitive dye, was certainly able to visualize the potential statuses of neurons and obtain physiological responses in both right and left visual cortex simultaneously by using multiple sensors for the first time. This accomplished multiplanar and multipoint measurement provides multidimensional information from different aspects. The light microsensors do not disturb the animal behavior. This implies that the imaging system can combine functional fluorescence imaging in the brain with behavioral experiments in a freely moving animal.