Control of interface functions in solid-state biosensors for stable detection of molecular recognition.

Significant progress has been achieved in the field of solid-state biosensors over the past 50 years. Various sensing devices with high-density integration and flexible configuration, as well as new applications for clinical diagnosis and healthcare, have been developed using blood, serum, and other body fluids such as sweat, tears, and saliva. A high-density array of ion-sensitive field effect transistors was developed by exploiting the advantages of advanced semiconductor technologies and commercialized in combination with an enzymatic primer extension reaction as a DNA sequencer in 2011. Different types of materials such as inorganic materials, metals, polymers, and biomolecules are mixed together on the surface of the gate while maintaining their own functions; therefore, compatibility among different materials has to be optimized so that the best detection performance of solid-state biosensors, including stability and reliability, is achieved as designed. Solid-state biosensors are suitable for the rapid, cost-effective, and noninvasive identification of biomarkers at various timepoints over the course of a disease.

Detection of Epidermal Growth Factor Receptor Expression in Breast Cancer Cell Lines Using an Ion-Sensitive Field-Effect Transistor in Combination with Enzymatic Chemical Signal Amplification.

A novel strategy for epidermal growth factor receptor (EGFR) detection using a cell-based field-effect transistor (FET) with enzymatic chemical signal amplification is proposed. Four human breast cancer cell lines [BT474, MDA-MB-231 (MM231), MDA-MB-468 (MM468), and MDA-MB-453 (MM453)] were used to compare the expression levels of EGFR. The cells were non-specifically captured on the surface of the gate of the FET, irrespective of their surface antigens. With this configuration, the heterogeneity of the cells would be analyzed using secondary antibodies conjugated to different kinds of enzymes. Four breast cancer cell lines with different levels of EGFR expression were captured on the respective surfaces of the extracellular matrix (ECM) gel-coated gates of the FETs. Glucose oxidase (GOx) was conjugated to the secondary antibody, and the output signals of the cell-based FETs changed depending on the expression levels of EGFR upon addition of glucose. The order of the expression levels of EGFR among the four cell lines, determined with the cell-based FETs, was consistent with the results of fluorescence detection determined by fluorescence-activated cell sorting (FACS). The cell-based FETs are advantageous for miniaturization and in massive parallel analyses of target molecules expressed on the membranes of cells and EVs, and their small size and cost effectiveness for cancer testing could enable their realization in a future liquid biopsy.

Detection of cell membrane proteins using ion-sensitive field effect transistors combined with chemical signal amplification.

The capture and detection of cells expressing a breast-cancer related membrane protein, namely a BT474 cell line expressing HER2, is demonstrated using ion-sensitive field effect transistors (ISFETs). BT474 cells were exposed to anti-HER2 antibodies and urease-conjugated secondary antibodies to induce chemical signal amplification by adding urea.

Molecular Interactions between an Enzyme and Its Inhibitor for Selective Detection of Limonene.

Researchers widely apply enzyme inhibition to chemicals such as pesticides, nerve gases, and anti-Alzheimer's drugs. However, application of enzyme inhibition to odorant sensors is less common because the corresponding reaction mechanisms have not yet been clarified in detail. In this study, we propose a new strategy for highly selective detection of odorant molecules by using an inhibitor-specific enzyme. As an example, we analyzed the selective interactions between acetylcholinesterase (AChE) and limonene─the major odorant of citrus and an AChE inhibitor─using molecular dynamics simulations. In these simulations, limonene was found to be captured at specific binding sites of AChE by modifying the binding site of acetylcholine (ACh), which induced inhibition of the catalytic activity of AChE toward ACh hydrolysis. We confirmed the simulation results by experiments using an ion-sensitive field-effect transistor, and the degree of inhibition of ACh hydrolysis depended on the limonene concentration. Accordingly, we quantitatively detected limonene at a detection limit of 5.7 µM. We furthermore distinguished the response signals to limonene from those to other odorants, such as pinene and perillic acid. Researchers will use our proposed odorant detection method for other odorant-enzyme combinations and applications of miniaturized odorant-sensing systems based on rapid testing.

Surface analysis of dental caries using a wireless pH sensor and Raman spectroscopy for chairside diagnosis.

A chairside tool for quantitative analysis of dental caries would improve clinical dental inspections. The wireless caries sensing tool with dental-explorer size has been developed comparing two sensing methods, Raman reading and pH reading for evaluating dental caries. The Raman spectra at 575 cm-1 and 960 cm-1 for in inorganic compounds, as well as 1450 cm-1 and 2940 cm-1 for organic compounds reinforced and supported the pH results. An Iridium/Iridium oxide (Ir/IrOx) pH sensing probe and wireless pH sensor (comprising an ESP8266 ESP-01 wireless module and ADS1115 analog digital converter) has been developed to quantitatively evaluate dental caries. All the operations of the wireless pH sensor were performed with a developed LabVIEW-based real-time data monitoring program. The slope and the linear fitting regression value (R2) of the wireless pH sensor using seven standards were -54.9 mV/pH and 0.999, respectively, showing high accuracy and stability for the pH measurements. The pH on the dental caries surface was measured with the wireless pH sensor, and the pH mapping results in the non-caries and caries areas were 6.9 and 5.7, respectively. The developed wireless pH sensor would be useful to understand the condition of dental caries and support dentists' inspection to remove only the caries part while keeping the non-caries structure.

Wafer-scalable chemical modification of amino groups on graphene biosensors.

Graphene's remarkable attributes make it suitable for application to biosensors for biomolecular recognition. Specific and precise target detection is realized by designing robust methods for immobilization of probe molecules, such as oligonucleotides, antibodies, receptors, and sugar chains, to a device surface. In this research, we developed a chemical modification method with a plasma treatment of amino groups on natural defects of graphene, which is compatible with a wafer-scalable semiconductor process, to prevent deterioration of the carrier mobility. The plasma treatment was optimized in terms of the efficiency of the amino radical generation, length of the mean free path, and reaction energy on graphene. The density of the modified amino groups on graphene was approximately 0.065 groups/nm2, and the change in the ΔId/ΔVg characteristic of the graphene field-effect transistor (FET) was negligible. DNA probes were then attached to the amino groups on the graphene FET. The target complementary DNA was detected at 1 nM after hybridization using the graphene FET devices. The plasma-assisted modification of the amino groups on the graphene surface was developed for immobilization of the DNA probes, and hybridization with the target DNA was demonstrated without deterioration of the carrier mobility.

Organic and inorganic mixed phase modification of a silver surface for functionalization with biomolecules and stabilization of electromotive force.

A solid-state potentiometric biosensor based on the organic and inorganic mixed phase modification of a silver surface is proposed. Stabilization of the electromotive force and functionalization with biomolecules on the sensing surface were simultaneously achieved using silver chloride chemically deposited with 1,3-diaminopropanetetraacetic acid ferric ammonium salt monohydrate and a self-assembled monolayer with oligonucleotide probes, respectively. The formation of silver chloride and adsorption of alkanethiol on the silver surface were confirmed with X-ray photoelectron spectroscopy. The resulting modified surface reduced the nonspecific binding of interfering biomolecules and achieved a high signal to noise ratio. The electromotive forces of the modified silver thin film electrodes were stable under constant chloride ion concentrations. Hybridization assays were performed to detect microRNA 146. The lower limit of detection was 0.1 pM because of the small standard deviation. The proposed biosensor could be useful as a disposable single-use sensor in medical fields such as liquid biopsies.

Liquid biopsy in combination with solid-state electrochemical sensors and nucleic acid amplification.

Solid-state electrochemical sensors are developing as a new platform for liquid biopsy, combining detection and analysis of nucleic acids with isothermal nucleic acid amplification reactions. For the detection of small nucleic acids such as cfDNA, ctDNA and miRNA in liquid biopsy, further development of highly sensitive and precise devices is required to transcend the barrier of classical detection technology. Electrochemical sensors based on engineering approaches are advantageous for miniaturization of systems and convenience of diagnosis, and the analysis of data obtained with these sensors dovetails with transition to an information society. This review briefly surveys the current development of electrochemical sensors combined with isothermal amplification for nucleic acid detection and predicts the future direction of research.

pH Mapping on Tooth Surfaces for Quantitative Caries Diagnosis Using Micro Ir/IrOx pH Sensor.

A quantitative diagnostic method for dental caries would improve oral health, which directly affects the quality of life. Here we describe the preparation and application of Ir/IrOx pH sensors, which are used to measure the surface pH of dental caries. The pH level is used as an indicator to distinguish between active and arrested caries. After a dentist visually inspected and defined 18 extracted dentinal caries at various positions as active or arrested caries, the surface pH values of sound and caries areas were directly measured with an Ir/IrOx pH sensor with a diameter of 300 µm as a dental explorer. The average pH values of the sound root, the arrested caries, and active caries were 6.85, 6.07, and 5.30, respectively. The pH obtained with an Ir/IrOx sensor was highly correlated with the inspection results by the dentist, indicating that the types of caries were successfully categorized. This caries testing technique using a micro Ir/IrOx pH sensor provides an accurate quantitative caries evaluation and has potential in clinical diagnosis.

Demonstration of thermo-sensitive tetra-gel with implication for facile and versatile platform for a new class of smart gels.

A tertiary branched poly(N-isopropylacrylamide) with controlled molecular weight, distribution and the end amino-functionalization (tetra-PNIPAAm-NH2) was studied for the ability to form a gel via in situ chain-end reaction with a counterpart tertiary branched poly(ethyleneglycol) bearing N-hydroxysuccinimide end groups (tetra-PEG-NHS), a well-documented class of building block to yield the tetra-gel. Some of these polymers, both comparable and distinct (relative to the counterpart) extended chain length pairs, provided a self-standing and macroscopically homogeneous gel, which was capable of undergoing thermo-sensitive and reversible change in hydration in line with the nature of PNIPAAm. Phantom network model based calculation indicated that a half molar fraction of the polymer chains in the network remained unreacted, revealing further room for optimizing the reaction condition. Since such tetra-PNIPAAm based motif can be readily tailored to a variety of other physicochemical stimuli-responsive analogues, our finding may give important insight into a platform for 'smart' tetra-gels with exceptional mechanical properties and potentially highly controllable molecular cut-off capability.

Electrochemical Biosensors Combined with Isothermal Amplification for Quantitative Detection of Nucleic Acids.

In recent years, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). The integration of isothermal amplification with electrical or electrochemical devices has enabled high-throughput nucleic acid-based assays with high sensitivity. We performed solid-phase rolling circle amplification (RCA) on the surface of a Au electrode, and detected RCA products in situ using chronocoulometry (CC) with [Ru (NH3)6]3+ as the signaling molecule. Detection sensitivity for DNA and a microRNA (miR-143) was 100 fM and 1 pM, respectively. Furthermore, we conducted potentiometric DNA detection using an ethidium ion (Et+)-selective electrode (Et+ISE) for real-time monitoring of isothermal DNA amplification by primer-generation RCA (PG-RCA). The Et+ISE potential enabled real-time monitoring of the PG-RCA reaction in the range of 10 nM-1 µM of initial target DNA. Devices based on these electrochemical techniques represent a new strategy for replacing conventional PCR for on-site detection of nucleic acids of viruses or microorganisms.

Real-time Monitoring and Detection of Primer Generation-Rolling Circle Amplification of DNA Using an Ethidium Ion-selective Electrode.

An electrochemical detection system for an isothermal DNA amplification method using an ion-selective electrode (ISE) was developed as a low-cost, simple and real-time monitoring system. The system is based on potentiometry using an ethidium ion (Et(+)) selective electrode that relies on monitoring DNA amplification by measuring potential changes in the reaction solution containing ethidium bromide (EtBr) as an intercalator to DNA. With progressing primer generation-rolling circle amplification (PG-RCA) under isothermal condition at 37°C, EtBr is bound to the newly formed DNA, resulting in a lowered free EtBr concentration in the sample solution. In this case, the Et(+) ISE potential allows real-time monitoring of the PG-RCA reaction in the range of 10 nM - 1 µM initial target DNA.

Measurement of Rapid Amiloride-Dependent pH Changes at the Cell Surface Using a Proton-Sensitive Field-Effect Transistor.

We present a novel method for the rapid measurement of pH fluxes at close proximity to the surface of the plasma membrane in mammalian cells using an ion-sensitive field-effect transistor (ISFET). In conjuction with an efficient continuous superfusion system, the ISFET sensor was capable of recording rapid changes in pH at the cells' surface induced by intervals of ammonia loading and unloading, even when using highly buffered solutions. Furthermore, the system was able to isolate physiologically relevant signals by not only detecting the transients caused by ammonia loading and unloading, but display steady-state signals as would be expected by a proton transport-mediated influence on the extracellular proton-gradient. Proof of concept was demonstrated through the use of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a small molecule inhibitor of sodium/hydrogen exchangers (NHE). As the primary transporter responsible for proton balance during cellular regulation of pH, non-electrogenic NHE transport is notoriously difficult to detect with traditional methods. Using the NHE positive cell lines, Chinese hamster ovary (CHO) cells and NHE3-reconstituted mouse skin fibroblasts (MSF), the sensor exhibited a significant response to EIPA inhibition, whereas NHE-deficient MSF cells were unaffected by application of the inhibitor.

Potentiometric responses of ion-selective microelectrode with bovine serum albumin adsorption.

There is a growing demand for an in situ measurement of local pH and ion concentrations in biological milieu to monitor ongoing process of bioreaction and bioresponse in real time. An ion-selective microelectrode can meet the requirements. However, the contact of the electrode with biological fluids induces biofouling by protein adsorption to result in a noise signal. Therefore, we investigated the relationship between the amount of nonspecific protein adsorption and the electrical signals in potentiometry by using ion-selective microelectrodes, namely silver/silver chloride (Ag/AgCl), iridium/iridium oxides (Ir/IrOx), and platinum/iridium oxides (Pt/IrOx). The microelectrodes reduced a potential change following the adsorption of bovine serum albumin (BSA) by comparison with the original metal microelectrodes without oxide layers. Suppression in the noise signal was attributed to the increased capacitance at the electrode/solution interface due to the formation of granulated metal oxide layer rather than a decrease in the amount of protein adsorbed. Ion sensitivity was maintained for Ir/IrOx against proton, but it was not for Ag/AgCl against chloride ion (Cl(-)), because of the interference of the equilibrium reaction by adsorbed BSA molecules on the electrode surface at<10(-2)M [Cl(-)] in the solution. The results open up the application of the Ir/IrOx microelectrode for measuring local pH in realistic dirty samples with a limited influence of electrode pollution by protein adsorption.

Electrical and electrochemical monitoring of nucleic Acid amplification.

Nucleic acid amplification is a gold standard technique for analyzing a tiny amount of nucleotides in molecular biology, clinical diagnostics, food safety, and environmental testing. Electrical and electrochemical monitoring of the amplification process draws attention over conventional optical methods because of the amenability toward point-of-care applications as there is a growing demand for nucleic acid sensing in situations outside the laboratory. A number of electrical and electrochemical techniques coupled with various amplification methods including isothermal amplification have been reported in the last 10 years. In this review, we highlight recent developments in the electrical and electrochemical monitoring of nucleic acid amplification.

Electrochemical label-free degranulation monitoring for in-situ evaluation of cellular function.

We fabricated a degranulation monitoring device, combining ion-sensitive field-effect transistor (ISFET) and microperfusion system. The electrical properties of ISFET were maintained even after immobilization of RBL-2H3 mast cells on the sensor. We successfully demonstrated in-situ monitoring of degranulation from stimulated RBL-2H3 cells by ionomycin. Potential change was induced by the release of acid-granule contents, which result in local pH decrease on the sensor under physiological conditions. This microdevice is expected to contribute as a platform technology for evaluating induced immune responses by chemical compounds.

Sensitive detection of microRNA by chronocoulometry and rolling circle amplification on a gold electrode.

We developed a quantitative detection scheme for nucleic acids, combining solid-phase rolling circle amplification and chronocoulometry (RCA-CC). A gold electrode was directly formed on a polystyrene substrate as a cost-effective and flexible biosensor for sensitive detection of microRNA (mir-143) in blood samples.

Thiolated 2-methacryloyloxyethyl phosphorylcholine for an antifouling biosensor platform.

We developed a new building block for a protein- and cell-repellant self-assembled monolayer (SAM) from 2-methacryloyloxyethyl phosphorylcholine (MPC) via a simple Michael-type addition to one mercapto group in alkanedithiol. The thiolated MPC can enable functionalization of a noble metal electrode to minimize noise signal in biosensing.