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Cardiovascular diseases (CVDs) are the leading causes of death worldwide. Despite the scientific and technological advances in healthcare, CVD mortality rate has not improved significantly. Medical intervention in diseases such as myocardial infarction (MI) rely on detection of protein biomarkers from blood, which require sophisticated laboratory based instruments that are costly, extremely complex and time-consuming. The general unaffordability and inaccessibility of the current medical diagnostics contribute towards fatalities and medical complications. We have devised a portable biosensor system to detect protein based biomarkers from a single drop of whole blood, in 5 minutes, without any additional sample pre-processing or extra reagents. The sensing methodology is based on electrical double layer (EDL) gated field effect transistor (FET) biosensor, in which an array of gold electrodes designed as extended gate structures, transduce immunological interaction of target protein to readable electrical signal. High electric field modulation of transistor drain current enables very high sensitivity, selectivity and wide dynamic range, in whole blood. Single drop of blood is dropped on the device and blood cells are gravitationally separated from blood plasma during 5 mins of incubation period, by rotating the device orientation to face downwards. A portable sensor measurement unit is designed to record the electrical characteristics of the sensor and display the test results in interfaced personal computer. We have demonstrated the detection of cardiac troponin I (cTnI) and brain natriuretic peptide (BNP) in 1X PBS and clinical whole blood samples. The sensor methodology has been developed to considerably reduce cost and assay complexity, with minimal user protocols. This whole blood assay can be performed at the convenience of homes, offices and hospitals, by anyone without prior training, in 5 minutes. Figure 1

In this research, the electrical double layer HEMT device is used to serve as the DNA biosensor. In this experiment, we try to use the DNA sequence of miRNA-126, which serve as a novel biomarkers of cardiovascular disease (CVD), to test the ability of the novel DNA sensors. Debye length has been a haunted problem among all the field effect transistor (FET) biosensor for years. As the Debye length is extremely low in high ionic strength solutions, the traditional FET may not be direct detect the biomolecular. The novel HEMT sensors using AlGaN/GaN can performs high sensitivity and great specificity in DNA sequences testing. The sensitivity can be down to the 1fM and the specificity measurement can separate the signals between two DNA sequences with two mismatch differences. Furthermore, the mechanism of the sensor structure has also been studied. Since the structure of the device has the separation between source drain channel and gate electrode, the comparison with different electrode area and gap distance has been done as well. With repeating electrical measurement confirming, the reusability can also be seen after the 95℃ dehybridization process. With the Gibbs free energy of the specific sequences, we can also predict the equilibrium reaction constant and the binding ratio of probe DNA with the target DNA. The comparison between the thermal dynamics with the experiment consequence has been demonstrated too. Figure 1

FET devices are extensively used for sensors, including gas sensors and biosensors. In contrast to traditional resistive devices, FET has transconductance gain, which can significantly improve the S/N ratio. Recently, more and more applications in biosensors combine microfluidic channels and the FET system. In FET-based biosensors, the wafer manufacturing process always requires a lot of cost, and most of the chip area is used for metal lines as interconnect. Miniaturized chip plays a key role in reducing the manufacturing cost. However, simultaneously integrating the sensor chip and microfluidic channels on one chip is challenging. In this study, a novel and robust packaging method has been developed, to effectively reduce the size of the sensor chip and thereby the cost. 1 mm x 1 mm FET sensor chip has been fabricated and embedded in epoxy. Photolithography and metal deposition were done followed by lift-off process for metal interconnect. The FET-embedded chip was passivated and covered by the microfluidic channel made of PDMS. Figure 1 shows the schematic of the system and the final package size is 1cm x 1cm. Electrical measurement is also proceeded with liquid sample to confirm the usability. Figure 1

Cardiovascular disease (CVD) is one of the major causes to claim millions of lives all across the globe every year. While the chances of CVD increase with age, it can also occur in any age group of people, both male and female. Despite all efforts and tremendous accomplishments in the precaution and treatment of CVD, its detection at an early stage is still a big challenge for scientists in this present modern world. Here, NT-pro BNP plays a crucial role in acute congestive heart failure since it is biologically inactive, binds better with its receptors and has a longer half-life than any other, making it an ideal biomarker. The detection method is based upon the ligand-receptor interaction, also called as immunoassay, which can be successfully detected with the use of High Electron Mobility Transistors (HEMTs). With its unique characteristic features like good electrical property, chemical stability, biocompatibility, thermal stability and low power consumption HEMT sensors also have been used in several other applications like ion-sensing, pH sensing, gas sensing, etc. Therefore, the detection method becomes simple, fast, convenient and cost effective. AlGaN/ GaN are the most promising candidates for HEMT sensor materials. A thin film of undoped Al0.25Ga0.75N layer (150 Å) is deposited onto the surface of 3 μm-thick undoped GaN buffer layer, which in turn is deposited on the top of Silicon substrate by metal-organic chemical vapor deposition (MOCVD) process. Using mesa ICP etching, active device area is formed. Then four different metals of various thicknesses, i.e. Ti, Al, Ni and Au, are deposited to make an ohmic contact and is annealed at an elevated temperature of 850ºC. Later, the third mask helps in making the final metal layer and the gate electrode used for immobilizing the receptor. Finally, the fourth mask lets us open some portion of the gate and the transistor region to allow liquid to cross through. The next step to sensor fabrication and device gate opening is making the surface ready for immobilization of receptor, or so called surface functionalization. The antibody disulfide bridges between the heavy chains are cleaved in the presence of mercaptoethylamine (MEA) and EDTA whereas the disulfide bridges between the heavy chain and the light chain remains unaffected. This leaves behind a number of disulfide bonds which can be bound onto gold surface easily because of the strong interactions between the native thiol group and the gold surface. Finally, the sensor measurement is performed with Agilent B1530 parameter analyzer. The figure 1 (c) represents the detection of NT- pro BNP at various concentrations. The graph shows the total charge accumulated against various concentrations of target proteins prepared in physiological environment (1X PBS). From the experiment performed, it can be observed that there is an increase in the change of charge with increase in the concentration of target proteins, which confirms the immobilization of antibody and ultimately the detection of target proteins. Moreover, the calibration curve for the sensor can be drawn by plotting change in the total charge against the concentrations with repeated testing of the device. Therefore, this method of detection is cheap, robust and time efficient making it also ideal for multi-sensor array and point-of-care diagnostics. Figure 1

Electric double layer was proved to modulate the channel of field-effect transistors, which is called electric-double-layer transistors (EDLTs) and have been reported previously. One of the unique property of the electric double layer is the much higher capacitance compared to that of silicon dioxide or other high dielectric constant materials. EDLTs are attractive not only because of its high capacitance, but also because of its other properties in solution, which is very different from conventional dielectric materials. In this study, we investigate the EDLTs using AlGaN/GaN HEMTs, with different designs of the devices and in solutions with different ionic strength. We find out that the channel conductivity is relevant to these parameters and the EDLT model becomes different from conventional FETs. By using the electric-double-layer AlGaN/GaN high electron mobility transistors, we successfully detect several proteins in physiological environment (1xPBS) with high ionic strength and in serum as well. Our devices and methods do not suffer from the charge screening effect, which is usually a critical issue for conventional FET-based biosensors. Direct detection of proteins in serum without any dilution or washing process can largely simplify the use of the FET-based biosensors for point-of-care or homecare. The results reveal future possible applications of the EDLTs.

Biomolecules such as DNA immobilized on metal electrodes and non-Faradaic current was measured in buffered solution. The metal-biomolecules-liquid-metal structure can be regarded as a liquid type of capacitor. The measurement of the capacitive current, which is affected by the ions, biomolecules, and liquid in the solutions. It is interesting to investigate the charging and discharging process in time domain, which brings important information of the dynamic behavior of the whole system. In this study, double-stranded DNA (dsDNA) was immobilized on gold electrodes and the capacitive current was measured in buffer solution with a pulse stimulus. Different relation time constants were revealed for various surface modifications. The transient current of the sensor was measured in de-ionized water and in 30 mM TE buffer before dsDNA immobilized metal electrode, and in 30 mM TE buffer after dsDNA immobilized on the metal electrode, followed by different concentrations of HIV-1 RT protein, ranging from 1 aM to 100fM. This technique shows ultra-high sensitivity of the protein detection (1 aM), indicating that this technique is promising for biosensor applications. Figure 1 shows the schematic of device for capacitive current measurements.

Hydrogen sulfide is a colorless, flammable and highly toxic gas with the characteristic foul odor of rotten eggs, often results from the bacterial breakdown of organic matters. Because of its toxicity, many personal safety gas detectors are set to alarm at 10ppm[i]. A number of sensors based on Schottky diode have been investigated as gas sensors since 1980s. In 2011, Professor Liu studied a Pt/AlGaN/GaN Schottky diode, which can exhibit ammonia detection limit lower than 35ppm NH3/air[ii]. There are also many papers research in H2S sensing with Schottky diode or metal oxide, most of their detection limit is higher than 10 ppm, expect one sensor made by thin film of CuO-SnO2, which can reach to 0.02ppm[iii]. The sensor studied in this paper contains four layers, which is showed in Figure 1 in detail. The Ohmic contact was formed by lift-off of e-beam evaporated Ti(200 Å)/Al(800Å)/ Ni(400Å)/Au(1000Å) and annealed at 850°C for 42 s under a flowing N2 ambient in a Premtek / ARTs 150. 100Å of Pt was deposited by e-beam evaporation to form Schottky contact as well as used as catalytic metal. Figure 2 shows three sets of Pt/GaN Schottky diode in different size and the size for the whole device is about 2×2mm. As the result of experiment, all of these three diodes are sensitive to H2S. Figure 3 shows real-time detection of 0.7ppm H2S in air. Figure 4 shows the relation between current change and concentration of H2S from 0.1-1ppm. All of the devices are tested in 200℃ and applied with 0.5ms width 1.5V pulse every second. With this Pt/GaN Schottky diode we can detect H2S down to 0.05pp. [i] Agency for Toxic Substances and Disease Registry (July 2006)."Toxicological Profile For Hydrogen Sulfide". p. 154. Retrieved 2012-06-20. [ii] T. Y. Chen, H. I. Chen, Y. J. Liu, C. C. Huang, C. S. Hsu, C. F. Chang, and W. C. Liu, “Ammonia sensing properties of a Pt/AlGaN/GaN Schottky diode,” IEEE Trans. Electron Devices, vol. 58, no. 5, pp. 1541–1547, May 2011. [iii] Jun Tamaki , Kengo Shimanoe, Yoshihiro Yamada, Yoshifumi Yamamoto, Morio Miura, and Noboru Yamazoe,” Dilute Hydrogen Sulfide Sensing Properties of Copper Oxide-Tin Oxide Thin Film Prepared by Low-Pressure Evaporation” Solid State Sensors and Actuators, 1997 International Conference Figure 1

Hydrogen peroxide is a metabolic by-product and a kind of stable reactive oxygen species (ROS). When the ROS levels increase, it may causes several harmful effects of ROS on the cell structure, such as damage of DNA and protein oxidation, and this is known as oxidative stress. The oxidative stress is also the important clinical indicators of cause of aging, Alzheimer disease, and kidney diseases. Now the common test method of oxidative stress is Enzyme-linked immunosorbent assay (ELISA). ELISA needs color giving dyes, complicated preparation of sample, and optical system, so it is very expensive and inconvenient. In our study, we present a high sensitivity hydrogen peroxide sensor with ultra-low detection limit. The sensor can directly test the sample and only need very small amount of sample. And because of the simple structural design and fabrication, the sensor can be used as a cheap, efficient, and portable sensor system. In the future, the novel hydrogen peroxide sensor has various applications in studying oxidative stress and detecting reactive oxygen species for cells. Furthermore, we can combine the PANI sensor with different enzymes to fabricate other highly sensitive sensors to detect diverse materials, such as glucose, lactic acid and cholesterin. In our study, the PANI layer and gold electrodes were deposited on silicon nitride substrate, and the PANI was applied to fabricate a thin film between two electrodes. Then the PANI layer was sultonated by propane sultone and modified with HRP. During the measurement, the sensor was operated at 100mV and different concentrations of hydrogen peroxide citrate buffer solutions (pH=5.4) were dropped on it. The current change was measured when the hydrogen peroxide reacted with the HRP Immobilized PANI thin film. We tested the hydrogen peroxide solution from 0.1 nm to 1mM. The HRP-modified resistive sensors based on n-alkylated polyaniline(PANI) detect hydrogen peroxide in solution with very high sensitivity, ultra-low limit, and short response time. The sensitivity is higher than that of other sensing methods, such as electrochemical sensors or transistor sensors. The detection limit of PANI sensor is 0.7 nM (Figure 1. a, b). It is three orders smaller than that of other common methods with detection limit around 1 μM. To the best of our knowledge, it is the lowest detection limit that has ever been reported. And We combine the hydrogen peroxide sensor with glucose oxidase to build up the glucose sensor. In summary, the hydrogen peroxide sensor can provide a more exact hydrogen peroxide concentration and quick detection. The simple process for the sensor fabrication also allows the sensor to be cheap, disposable and combinable with other sensors to build up sensing system. This work was partially supported by National Science Council grant (No.99B20495A & 101-2221-E-007-102-MY3) and by the research grant (100N2049E1) at National Tsing Hua University.

Paper is an important material in our daily lives. Paper is cheap, environmentally friendly, light, flexible and widespread all over the world. Paper-based sensors are attractive because they are cheap, portable, accessible everywhere, and easily destroyed after being used. Paper-based sensors have been widely used in food safety control, clinical test, and environmental detection. The fiber structure in paper, which can act like a tunnel for microfluidic liquid transport, plays an important role in paper-based sensors by providing a platform for chemical reaction of analytes. However, conventional paper-based biosensors, which usually involve colorimetry, has the limitation in sensitivity and detection limit. Besides, the quantitative analysis of optical signals usually requires expensive optical detection system. In sharp contrast, most electronic sensors can be fabricated in very low cost and display with a simple device. In our study, we present a novel idea, which turns regular paper into conductive by combining paper and conducting polymer together. By integrating this conducting paper with different sensor technologies, various sensors can be achieved with high sensitivity and accuracy, which can be attributed to the great biocompatibility of the conducting paper. Only small amount of sample is required for testing with these sensors. Because of the simple structural design and fabrication, the conducting paper can be fabricated as a cheap, efficient, and portable electronic biosensor. Previous research has shown the HRP-modified n-alkylated polyaniline (PANI) can detect hydrogen peroxide in buffer solution with a very high sensitivity, ultra-low detection limit, and very short response time. During fabrication, the PANI thin film may sometime suffer from cracks after baking the PANI layer on the silicon nitride substrate due to different thermal expansion coefficients between PANI and silicon nitride. By using a filter paper as the substitute for hard substrate, the mechanical stress of PANI after baking can be largely reduced. Figure 1 shows the schematic of the conducting paper. Propane sultone was doped in PANI. The PANI solution was then dropped on the filter paper and silver electrode is then coated on the back of the paper to contact with the PANI, extending to the front side, making it easy for probing. The current was measured from 0-1V. Figure 2 shows the current-voltage characteristics of the conducting paper. Figure 3(a) shows that the PANI layer is tightly merged with the filter paper Figure 3(b) shows the surface morphology of PANI on the paper. In summary, the conducting paper made of PANi can not only achieve electronic paper-based devices, but can also realized electronic paper-based biosensors which can provide better sensitivity and lower cost, compared to the conventional paper-based sensors. Combining it with different technologies and enzymes, it can be used to detect several kinds of analytes. The portability and flexibility of conducting paper also make it more convenient to use. Because the fabrication process is quite simple and cheap, it can be widely promoted commercially in the future. This work was partially supported by National Science Council grant (No.99B20495A & 101-2221-E-007-102-MY3) and by the research grant (100N2049E1) at National Tsing Hua University.

Hydrogen peroxide attracts a great interest due to its important role in food, pharmaceutical, and clinical applications. Hydrogen peroxide is also a by-product in many enzyme catalytic reactions, such as glucose oxidase, lactate oxidase, cholesterol oxidase, alcohol oxidase, urate oxidase, aldehyde oxidase, and oxalate oxidase, which are implemented to detect glucose, lactic acid, cholesterol, ethanol, urea, formaldehyde, and oxalate, respectively. These biomolecules are significant markers in many biologically metabolic reactions. In our study, we present a cholesterol sensor based on a high sensitivity hydrogen peroxide sensor with ultra-low detection limit. The sensor can directly test the sample and only need very small amount of sample. And because of the simple structural design and fabrication, the sensor can be used as a cheap, efficient, and portable sensor system. The HRP-modified resistive sensors based on n-alkylated polyaniline(PANI) detect hydrogen peroxide in solution with very high sensitivity, ultra-low limit, and short response time. The sensitivity is higher than that of other sensing methods, such as electrochemical sensors or transistor sensors. The detection limit of PANI sensor is 0.7 nM. It is three orders smaller than that of other common methods with detection limit around 1 μM. To the best of our knowledge, it is the lowest detection limit that has ever been reported. And we combine the hydrogen peroxide sensor with cholesterol oxidase to build up the cholesterol sensor. In our study, the PANI layer and gold electrodes were deposited on silicon nitride substrate, and the PANI was applied to fabricate a thin film between two electrodes. Then the PANI layer was sultonated by propane sultone and modified with HRP. After that, the device was combined with dialysis membrane which was modified with cholesterol oxidase. During the measurement, the sensor was operated at 100mV and different concentrations of cholesterol PBS solutions (pH=7.0) were dropped on it. The cholesterol reacted with the cholesterol oxidase and created hydrogen peroxide on the PANI thin film (Figure 1). The current change was measured when the hydrogen peroxide reacted with the HRP immobilized PANI thin film. According to regular cholesterol level in human blood, we tested the cholesterol solution from 100 mg/dl to 400 mg/dl ,and we get a very good linear result (Figure 2). In summary, the cholesterol sensor can provide a more exact cholesterol concentration detection. The simple process for the sensor fabrication also allows the sensor to be cheap, disposable and combinable with other sensors to build up sensing system. This work was partially supported by National Science Council grant (No.99B20495A & 101-2221-E-007-102-MY3) and by the research grant (100N2049E1) at National Tsing Hua University.

Recently, more and more studies indicate that free radical is the cause of various diseases such as aging, cancer, neurodegenerative diseases and cardiovascular diseases because of uncontrolled oxidation of lipids, protein and DNA in biological system. Hydroxyl radical (OH°) is the highest oxidized species in free radicals. So far, most techniques for hydroxyl radical detection are sophisticated and expensive. Therefore, a simple and cheap method for hydroxyl radical detection is very necessary. Polyaniline (PANI) has been used as the active layer for different type of sensors because of its good electronic conductivity. The conductive polyaniline can neutralize free radicals by donating electrons and thereby changing their oxidation state from emeraldine to pernigraniline. In here, we used a simple and cheap polyaniline microchip to detect hydroxyl radical generated from Fenton reaction. The result shows significant current (or conductance) change. Figure 1 (a) and (b) show the schematic of hydroxyl radical sensor and the top-view of the devices, respectively. The sensor consists of two metal electrodes made by 200 Å Ti and 1000 Å Au deposited with an e-beam evaporator on a Si3N4/Si substrate. The length and the width of the Au electrodes are 500 μm and 100 μm, respectively. The gap between the two metal electrodes is 10 μm. 0.3 g polyaniline emeraldine base powder was dissolved in 5 mL dimethyl sulfoxide (DMSO) with stirring for 6 h. The polyaniline solution was then mixed with the same volume of 0.01 M NaOH. Use centrifuge to separate two different phase in the solution and pipet out the upper layer of the solution. Propane sultone was added into the polyaniline solution with the ratio 3:7 and stood for 6~8 hr over 30 degree Celsius. Also use centrifuge to separate two different phase in the solution and pipet out the upper layer of the solution. 2 μL of the remaining solution was coated on a chip and baked for 2.5 hr. The hydroxyl radical was generated from Fenton reaction as shown below: Fe2+ + H2O2 → Fe3+ + OH°+ OH- Figure 2 (a) shows significant current (or conductance) decrease approximately 84 μA of hydroxyl radical sensor after dropping hydrogen peroxide. It indicates that hydroxyl radical reacted with polyaniline and stole electron from polyaniline inducing current decrease. The current of the sensor was measured at a constant bias of 0.1 V at room temperature by using Agilent B1500 parameter analyzer with the polyaniline layer exposed. The interval time between any two measurement points is 1 minute. The final concentration of ferrous ion and hydrogen peroxide are 25 mM and 100 mM, respectively. Both two solutions were prepared in citrate phosphate buffer solution. Figure 2 (b) shows the current of sensor after dropping the hydrogen peroxide without ferrous ion. The current doesn’t change significantly. It indicates that the sensor doesn’t react with the hydrogen peroxide. Therefore, we can verify that the signal in figure 2 (a) is from hydroxyl radical, not from hydrogen peroxide. In summary, we develop a simple and cheap sensor to detect hydroxyl radical. The sensor shows significant conductance change when reacting with hydroxyl radical. We will further test different concentration of hydroxyl radical and confirm the mechanism in the future. This work was partially supported by National Science Council grant (101-2221-E-007-102-MY3) and by the research grant (101N7047E1) at National Tsing Hua University.

Variation of temperature, pressure, and pH values has been demonstrated for affecting the stability of AlGaN/GaN HEMTs sensors. In addition to the factors above mentioned, we found out other factors which are related to the stability of AlGaN/GaN HEMTs sensors. In this study, we found out the current variation of AlGaN/GaN HEMTs without gate metal sensors is inversely proportional to the viscosity of liquids. We also found out that dipole moment of liquids is related to the current variation of AlGaN/GaN HEMTs without gate metal sensors.

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