DESCRIPTION

SENSORS USING AlGaN/GaN HIGH ELECTRON MOBILITY TRANSISTORS

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the benefit of U.S. Provisional Application Serial No. 60/973,302, filed September 18, 2007, claims the benefit of U.S. Provisional Application Serial No. 60/975,907, filed September 28, 2007, and U.S. Provisional Application Serial No. 60/982,310, filed October 24, 2007, all of which are hereby incorporated by reference herein in their entirety, including any figures, tables, or drawings.

BACKGROUND OF THE INVENTION Chemical sensors can be used to analyze a wide variety of environmental and bodily gases, aerosols and fluids for properties of interest. For example, exhaled breath condensate (EBC) is widely known to be a diagnostically important bodily fluid that can be safely collected. In particular, the breath from deep within the lungs (alveolar gas) is in equilibrium with the blood, and therefore the concentrations of molecules present in the breath is highly correlated with those found in the blood at any given time. Analysis of molecules in exhaled breath condensate is a promising method that can provide information on the metabolic state of the human body, including certain signs of cancer, respiratory disease and liver and kidney function. Several different analysis methods including gas chromatography (GC), chemiluminescence, selected ion flow tube (SIFT), and mass spectroscopy (MS) have been used to measure different exhaled biomarkers, including hydrogen peroxide, nitrogen oxide, aldehydes, and ammonia. However, these methods show vary significantly in sensitivity.

Another example of sensing application using body fluid is detecting breast cancer with saliva. The mortality rate in breast cancer patients can be reduced by increasing the frequency of screening. The overwhelming majority of patients are screened for breast cancer by mammography. This procedure involves a high cost to

the patient. Moreover, the use of invasive radiation limits the frequency of screening. Recent evidence suggests that salivary testing for markers of breast cancer may be used in conjunction with mammography. Saliva based diagnostics for the protein c- erbB-2, a prognostic breast marker assayed in tissue biopsies of women diagnosed with malignant tumors, has shown tremendous potential. Soluble fragments of the c- erbB-2 oncogene and the cancer antigen 15-3 were found to be significantly higher in the saliva of women who had breast cancer than in those patients with benign tumors. Another recent study concluded that epidermal growth factor (EGF) is a promising marker in saliva for breast cancer detection. Pilot studies indicate that the saliva test is both sensitive and reliable and is potentially useful in initial detection and follow-up screening for breast cancer. However, currently saliva samples are typically obtained from a patient in a dentist's office then sent to a testing lab; it typically takes a few days to get the test results.

To fully realize the potentials of sensors for environmental, health related, chemical and biomedical applications, technologies are needed that will enable easy, sensitive, and specific detection of chemical or biomolecules at home or elsewhere. It is also desirable that a testing device allows concomitant wireless data transmission into preprogrammed destinations, such as transmitting breast cancer testing results to a doctor or clinic. If inexpensive technologies that can detect and wirelessly transmit testing results for environmental, health related, chemical and biomedical applications can be developed, early diagnosis of cancers or disease can significantly lower mortality and the cost of health care. Real-time wireless remote sensing for chemicals in the environment may reduce the incidence of disasters by alert to a chemical hazard.

BRIEF SUMMARY

AlGaN/GaN high electron mobility transistors (HEMTs) are a key component of the sensors according to embodiments of the invention. AlGaN/GaN HEMTs with different surface functionality comprise sensors to detect various molecules, including biomarkers, of interest in different bodily fluid samples. For example, embodiments of the invention are directed to different surface functionalized AlGaN/GaN HEMT based sensors to detect prostate cancer, breast cancer, pH, mercury and/or copper,

glucose, and markers for acute kidney injury or renal failure in samples of exhaled breath condensate, saliva, urine, blood, or other fluids. The devices according to embodiments of the invention can also wirelessly transmit results of analysis employing the sensors to facilitate rapid analysis of test results. One embodiment of the invention is a device for detecting breast cancer that includes a gold-gated AlGaN/GaN HEMT functionalized with a breast cancer antibody as the detector. A method for detection of breast cancer in a saliva sample is disclosed.

A second embodiment of the invention is a device for detection of heavy metals that includes a gold-gated AlGaN/GaN HEMT functionalized with a chelating agent as the detector. A method for detection of heavy metals in solution comprises analyzing a sample with a detector functionalized with a chelating agent such as thioglycolic acid (HSCH ₂ COOH), cysteamine (NH ₂ CH ₂ CH ₂ SH), 1,2-ethanedithiol (HSCH ₂ CH ₂ SH), dimercaprol (BAL), diaminoethanetetraacetic acid (EDTA), 2,3-bis- sulfanyϊbutanedioic acid (DMSA), or 2,3-dimercapto-l-propanesulfonic acid

(DMPS).

A third embodiment of the invention is a device for detecting changes in pH that includes an AlGaN/GaN high electron mobility transmitter having a gate dielectric coating, for example a thin Sc ₂ O ₃ layer, as the detector. The device can include a cooling device to obtain exhaled breath condensates for testing. A method for detecting the pH of exhaled breath condensate is disclosed.

A fourth embodiment is a device for detecting prostate cancer using a detector formed via carboxylate succinimdyl ester bound prostate specific antigen (PSA) antibodies linked to thioglycolic acid immobilized on the AlGaN/GaN HEMT Au- coated gate. A method of detecting prostate cancer by analysis of PSA in solution is disclosed.

A fifth embodiment is a device for detecting acute kidney injury or renal failure where a gold-gated AlGaN/GaN HEMT functionalized with highly specific KIM-I antibodies through a self-assembled monolayer of thioglycolic acid acts as the detector. A method to detect KIM-I in solution is disclosed.

A sixth embodiment is a device for detecting glucose in exhaled breath condensate having a detector where an AlGaN/GaN HEMT has a gate that comprises

a nanorod array selectively grown on the gate area that immobilizes glucose oxidase (GOx). The nanorods may be, for example, metal oxide and nitride based nanorods. A method to detect glucose in exhaled breath condensate is disclosed.

BRIEF DESCRIPTION OF THE FIGURES

Figures IA - ID show the chemical structures for BAL, EDTA, DMSA, and DMPS, respectively.

Figure 2 shows a schematic of an exemplary embodiment of an AlGaN/GaN HEMT sensor of the present disclosure. Figure 3A shows a scanning electron microscope (SEM) image of an exemplary gateless HEMT.

Figure 3B shows a schematic diagram of an exemplary HEMT with a non- native oxide as a gate dielectric layer.

Figure 4 A shows the effects of EBC exposure, in the form of multiple exhaled breaths (< 1 sec), on the current change.

Figure 4B shows the effects of ventilation strength on the HEMT current for 5 seconds.

Figures 5A and 5B are photographs of contact angle of water drop on the surface of bare Au (Figure 5A) and thioglycolic acid functionalized Au (Figure 5B). Figure 6 illustrates changes in HEMT drain-source current for bare Au-gate and Au-gate with thioglycolic acid functionalization exposed to 10 ^"5 M Hg ²⁺ ion solutions when using an AlGaN/GaN HEMT sensor of the invention.

Figure 7 A shows time dependent response of the drain current as a function of Hg ^"" , Cιf ^τ , Pb ^" ^ ion concentrations for bare Au-gate AlGaN/GaN HEMT sensor. Figure 7B shows time dependent response of the drain current as a function of

Hg ^~ \ Cu' , Pb ^" ion concentrations for thioglycolic acid functionalized Au-gate AlGaN/GaN HEMT sensor.

Figure 8A shows drain current changes in response to Hg ²⁺ and Cu ^2* ions as a function of the ion concentration for the bare Au-gate AlGaN/GaN HEMT sensor. Figure 8B shows drain current changes in response to Hg ^~~ and CiT ⁺ ions as a function of the ion concentration for the thioglycolic acid functionalized Au-gate AlGaN/GaN HEMT sensor.

Figure 9 shows a plan view photograph of a multiple cell AlGaN/GaN HEMT sensors.

Figure 10 shows time dependent change in the drain current in response to Na ⁺ and Mg ^* with bare Au-gate and thioglycolic acid functionalized Au-gate HEMT sensor of the invention.

Figure HA shows recyclability for the bare Au-gate.

Figure HA shows recyclability for the thioglycolic acid functionalized Au- gate surface.

Figure 12A shows a plan view photomicrograph of a completed HEMT device of the invention with a 5-nm Au film in the gate region.

Figure 12B shows a schematic of an AlGaN/GaN HEMT of the invention, where the Au-coated gate area was functionalized with PSA antibody on thioglycolic acid.

Figure 13A shows I-V characteristics of an AlGaN/GaN HEMT sensor of the invention before and after PSA incubation.

Figure 13B shows drain current over time for PSA when sequentially exposed to PBS, BSA, and PSA when using an AlGaN/GaN HEMT sensor of the invention.

Figure 14 A shows drain current over time for PSA from 10 pg/ml to 1 ng/ml when using an AlGaN/GaN HEMT sensor of the invention. Figure 14B is a graphical illustration of change of source and drain current versus different concentrations from 10 pg/ml to 1 μg/ml of PSA using an AlGaN/GaN HEMT sensor of the invention.

Figure 15 shows the change in current in HEMT with a gate dielectric of a non-native oxide at fixed source-drain bias of 0.25 V with pH from 3-10. FIG 16 shows the change in current in HEMT with a gate dielectric of a non- native oxide at fixed source-drain bias of 0.25 V with pH from 7-8.

Figure 17A shows a plan view photomicrograph of a completed device with a 5-nm Au film on the gate region.

Figure 17B shows a schematic device cross section where the Au-coated gate area was functionalized with KIM-I antibody on thioglycolic acid.

Figure 18 shows I _DS -V _DS characteristics of HEMT in both PBS buffer and lOOng/ml KIM-I .

Figure 19 shows time dependent current signal when exposing the HEMT to lng/ml and lOng/ml KlM-I in PBS buffer.

Figure 20 shows the current change in HEMT as a function of KIM-I concentration. Figure 21 shows field emission SEM images of ZnO nanorod arrays grown on the Si substrate spin coated with different size precursors (Preparation time of ZnO nanoparticle precursors from (a) to (d): 0.5, 1, 1.5, and 2 h).

Figure 22 shows (a) Schematic of ZnO nanorod gated AlGaN/GaN HEMT; (b) SEM image of ZnO nanorod gated AlGaN/GaN HEMT. Upper right inset shows HRTEM image of a ZnO nanorod arrays grown on the gate area with different scales.

Figure 23 shows plot of drain current versus time with successive exposure of glucose from 500 pM to 125 μM in 10 mM phosphate buffer saline with a pH value of 7.4.

Figure 24 shows plot of change of drain current as a function of glucose concentrations from 500 pM to 125 μM in 10 mM phosphate buffer saline with a pH value of 7.4.

DETAILED DISCLOSURE Group III-N based wide bandgap semiconductors are used as sensitive chemical sensors, especially when made with piezoelectric materials. GaN/ AlGaN high electron mobility transistors (HEMTs) allow sensors that form a high density electron sheet carrier concentration channel induced by piezoelectric polarization of the strained AlGaN layer and spontaneous polarization of the different ionic strength between the GaN and AlGaN layer. The conducting 2-dimensional electron gas (2DEG) channel of GaN/ AlGaN based HEMTs is very close to the surface and extremely sensitive to the ambient environment, allowing enhanced detection sensitivity.

Gateless HEMT structures can distinguish liquids with different polarities and can quantitatively measure pH over a broad range. The sensing mechanism for chemical adsorbates in piezoelectric materials originates from compensation of the polarization induced bound surface charge by interaction with the polar molecules in liquids. In gateless aluminum gallium nitride/gallium nitride (AlGaN/GaN)

heterostracture transistors, the native oxide on the nitride surface is responsible for the pH sensitivity of the response to electrolyte solutions. By coating a thin non-native metal oxide, for example Sc ₂ O ₃ , on the gate sensing area, referred to as a gate dielectric layer in embodiments of the invention, more sensitive and reproducible hydronium ion, H ₃ O ⁺ (pH) sensing is achieved. Usually, it is difficult to control the compositions and thickness of native oxides. For embodiments of the invention, a metal oxide such as Sc ₂ O ₃ is grown by a molecule beam epitaxy system with excellent composition and thickness control. Other gate dielectric layers, such as metal nitrides, can be used rather than metal oxide dielectric layers. The pH response of oxide/nitride interface can be modeled in terms of formation of hydroxyl groups that lead to a hydronium ion concentration (pH) dependent net surface change with a resulting change in voltage drop at the semiconductor/liquid interface.

Biologically modified field effect transistors (bioFETs), either at conventional or nano-dimensions, can directly detect biochemical interactions in aqueous solutions for a wide variety of biosensing applications. To enhance the practicality of bioFETs, a device according to embodiments of the invention is sensitive to biochemical interactions on its surface that is functionalized to probe specific biochemical interactions. In one embodiment, the device is stable in aqueous solutions across a range of pH and salt concentrations. In other embodiments, the gate region of the device is covered with biological probes, which are used as receptor sites for molecules of interest. The conductance of the device changes as reaction occurs between these probes and appropriate species in solution.

GaN-based wide energy bandgap semiconductor material systems are extremely chemically stable; this stability is minimally degraded by adsorbed cells. The bond between Ga and N is ionic and proteins easily attach to the GaN surface.

This is an important factor for preparation of a sensitive biosensor having a useful lifetime.

HEMT sensors can be used for detecting gases, ions, pH values, proteins, and DNA with good selectivity by modification of the surface in the gate region of the HEMT. HEMT structures can be used in microwave power amplifiers as well as gas and liquid sensors because of their high 2DEG mobility and saturation velocity.

In embodiments of the invention, a 2-dimensional electron gas (2DEG) at the interface of AlGaN/GaN heterostractures is formed through the hetero-junction of AlGaN and GaN, which have different bandgaps. The 2DEG channel is connected to an Ohmic-type source and drain contacts. The source-drain current is modulated by a third contact, a Schottky-type gate, on the top of the 2DEG channel. For sensing applications, the third contact is affected by the sensing environment, i.e. the sensing targets (substrates) changes the charges on the gate region and the behavior of the gate. When analytes accumulate on the gate area, the net charge on the HEMT surface is changed. The net surface charge alters the 2DEG concentration. This electrical detection technique is simple, fast, and convenient. The detecting signal from the gate is amplified through the drain-source current, making the sensor very sensitive. The electric signal can be easily quantified, recorded and transmit, unlike a fluorescence detection method that needs human inspection and is difficult to be precisely quantify and transmit. One shortcoming of HEMT sensors has been lack of selectivity to different analytes due to the chemical inertness of the HEMT surface. Sensor devices according to the embodiments of the invention solve this sensitivity problem by functionalization of the gate surface with detecting receptors. The sensor devices can be used with a variety of solutions having environmental and bodily origins, including saliva, urine, blood, breath (including exhaled breath condensates) and other solutions. For example, in two embodiments of the invention, mercury or cancer detection is improved. Additionally, sensors according to embodiments of the invention can be re-used, without substantial diminishment of efficacy.

According to an embodiment of the subject invention, real-time detection of the pH of exhaled breath uses a breathing tube and ice bath with an AlGaN/GaN

HEMT. The breathing tube samples exhaled breath and the ice bath condenses the sample that is applied to the AlGaN/GaN HEMT.

In one embodiment, the device may include an AlGaN/GaN HEMT that is operably coupled to a thermal electric cooling device, which condenses exhaled breath samples. The thermal water vapor and volatile organic compounds from the exhaled breath condensate change the surface charge on the HEMT to which it is applied, thus changing the current flowing in the HEMT device for a fixed applied

bias voltage. In one embodiment, an exhaled breath condensate (EBC) biosensor of the present disclosure can be handheld, low in cost, and capable of real time detection without consumable carrier gases.

In one embodiment of the invention, a saliva based breast cancer detector is functionalized in the gate region with chemicals that can bind commercially available breast cancer markers. The gate region of the HEMT can be of a few nanometers to a few millimeters in size. An array of the HEMT sensors can be fabricated on a single chip. Each HEMT can be functionalized with a different cancer marker. The sensing thereby consists of a set of testing results from a series of different bio-markers. Simultaneous breast cancer detections with different bio-markers can increase the accuracy of the cancer detection.

Since the surface of AlGaN is extremely inert and difficult to oxidize, a thin layer of gold of about 5nm can be used as an intermediate layer between AlGaN and the bio-markers used in embodiments of the invention. A molecule containing a thiol group can be immobilized on the Au surface by a Au-S bond. Another functional group can bind with a biomarker; such functional group can be alcohol, aldehyde, carboxylic acid, phosphate or amine groups, for example. The immobilized, bio- markers can bind with breast cancer cells. In one embodiment an Au-gated GaN/ AlGaN HEMT comprises a saliva based sensor for the detection of breast cancer.

In some embodiments, sensors comprise chemical adsorbates on AlGaN/GaN HEMTs where detection originates from compensating or inducing charges at the AlGaN/GaN interface due to the polar molecules in the liquids bonded to the AlGaN/GaN surface. In some embodiments, the device is functionalized at the AlGaN/GaN/HEMT surface having an Au-coated gate region by different specialized chemicals.

According to an embodiment of the invention, thioglycolic acid can be used to assist in functionalizing an AlGaN/GaN HEMT sensor. For example, a self- assembled monolayer of thioglycolic acid can be adsorbed onto the Au-gate due to interaction between gold and the thiol-group. Following placement of the thioglycolic acid on the sensor surface, specific functionality of interest may be conjugated to the surface, where functionality acts as a receptor for a substrate to be sensed.

According to embodiments of the invention, a sensor can include a synthetic or natural compound as a receptor with the ability to associate with a desired substrate, target molecule that can be a biomarker. Such receptors include naturally occurring and/or synthetic compounds that display high specificity and sensitivity to a substrate that is one or more target chemicals and/or biological molecules of interest.

Suitable compounds include, but are not limited to, antibodies, proteins, and aptamers which can associate with a biomarker. The term "biomarker" refers to a biochemical in the body with a particular molecular trait that makes it useful for diagnosing a condition, disorder, or disease and for measuring or indicating the effects or progress of a condition, disorder, or disease. Antibodies are protein molecules that are composed of equal numbers of heavy and light polypeptide amino acid chains held together with disulfide bonds. These highly specialized proteins are able to recognize and bind selectively certain types of antigen molecules at receptor sites. In embodiments of the invention, a sensor employs antibodies to detect specific antigens. In an embodiment of the invention, the chemistry of the system occurs along a conductive layer, for example, a gold layer. The conductive layer support the propagation of a high frequency test signal and are capable of binding the substrate, which is a desired antigen or other analyte. In one embodiment, thioglycolic acid bonds the Au layer to antibodies for breast cancer antigens including (but not limited to) EGF, c-erbB-2 and CAl 5-3 in saliva, where the thiogylcolic acid forms a self- assembled monolayer on the gold surfaces. Upon binding of the immobilized antibody to an antigen, the gate potential of the HEMTs changes, resulting in a current change in the HEMT at fixed bias voltage. This change allows quantification of the amount of the target, for example a cancer biomarker, in the solution. In an embodiment of the invention, functionalization is the gold surface of the gate with thioglycolic acid coupling kidney injury molecule- 1 (KIM-I) antibodies to the gold surface. When in the presence of KIM-I, the gate potential of the HEMTs changes, resulting in a current change in the HEMT at fixed bias voltage. This change can be used to quantify the amount of the KIM-I biomarker present in the solution.

One embodiment of the invention is a portable or hand-held saliva based breast cancer sensor. Other embodiments of the invention are sensor to analyze other

bodily fluids or excretions such as breath, urine or blood. Advantages of the sensors include fast response time for results, portability and low cost. In one embodiment, a chemical sensor array can be integrated with wireless communication circuits for remote sensor applications. For example, a digital signal cancer detector can wirelessly send the testing results directly to a user's doctor.

In another embodiment of the invention, the sensor is used to detect heavy metals. Heavy metal detection involves selecting a powerful receptor and densely coating it on the surface of HEMTs. Hg ²⁺ , Cu ⁺² and Pb ²⁺ detection is based on an effective medical approach for heavy metal poisoning, so called "Chelation Therapy." One embodiment is to a method in which chelating agents remove heavy metal ions from a sample, where chelating ligands and metal ions bind to form metal complexes, normally called "chelation." A strong chelating agent is dimercaprol (BAL), which contains two thiol groups capable of reacting with arsenic, lead and mercury. Other widely used chelating agents include diaminoethanetetraacetic acid (EDTA), 2,3-bis- sulfanylbutanedioic acid (DMSA), and 2,3-dimercapto-l-propanesulfonic acid

(DMPS). Figures IA - ID show the chemical structures for BAL, EDTA, DMSA, and DMPS, respectively.

In one embodiment of the invention, Hg ⁺ , Cu ⁺ or Pb ⁺ is detected when chelating receptors immobilize the metal on the HEMT surface. The surface of AlGaN can have a thin layer (~5nm) of gold between the AlGaN surface and the chelating receptors. Gold permits deposition of any chelating agent comprising a thiol group, on the surface through Au-S bonding. The thiol, amine, and carboxyl groups of the bound chelating agents bind heavy metal ions to the surface of the HEMT. Figure 2 shows a schematic of an exemplary embodiment of an AlGaN/GaN

HEMT sensor according to an embodiment of the invention. The functionalization is an Au-coated gate area with thioglycolic acid, HSCH ₂ COOH, for Hg(II) detection. A self assembled monolayer of thioglycolic acid molecules is adsorbed onto the gold gate by a S-Au bond between the gold surface and thiol-group. The immobilized carboxyl groups function as receptors to capture Hg ²⁺ , Cu ^{^2} or Pb ²⁺ ions. Alternative binding groups are derived from cysteamine (NH ₂ CH2CH ₂ SH) or 1,2-ethanedithiol (HSCH ₂ CH ₂ SH), for example.

In one embodiment, the gold-gated region is functionalized with chelating receptors immobilized on the HEMT surface, such as BAL, EDTA, DMSA, and DMPS. One portion of the chelating agent binds to the Au surface and the other portions chelates with heavy metals, such as Hg ⁺ , Cu ⁺ or Pb ⁺ . The charge of the metal ions affects the gate potential of HEMTs. The change in current in the HEMT at fixed bias voltage allows quantification of the amount of the heavy metal ions in a solution. In one embodiment, the device is a portable or hand-held trace heavy-metal sensor for environmental and health related applications. The sensor can detect heavy metals in aqueous solution including breath condensate, urine or blood. Advantages of the sensing device include fast response time, portability and low cost. In one embodiment, a heavy metal detector can be used as a wireless based sensor to transmit a digital signal of the test results directly to a recipient.

Another embodiment of the invention is a pH meter for fluids such as breath, saliva, urine or blood. Gates can be functionalized with noble metal oxides for detecting proton and hydroxide ions. In one embodiment, a Sc ₂ O ₃ gate dielectric is placed on AlGaN/GaN HEMTs to provide high sensitivity for detecting changes in pH of electrolyte solutions. HEMTs with Sc ₂ O ₃ exhibit a linear change in current between a pH range of 3 to 10 of 37μA/pH. The HEMT pH sensors are stable with a resolution of < 0.1 pH over the entire pH range. The HEMTs can be used to monitor solution pH changes between 7 and 8, a range of interest for testing human blood.

Figure 3A shows a scanning electron microscope (SEM) image of a HEMT with a gate dielectric layer (oxide). Figure 3B shows a schematic diagram of a HEMT with a gate dielectric layer (oxide). Figure 4A shows the effects of EBC exposure, in the form of multiple exhaled breaths (< 1 second), on the current change. Figure 4B shows the effects of ventilation strength on the HEMT current for 5 seconds.

In other embodiments of the invention, a nanorod gated AlGaN/GaN HEMT is a detector for glucose. The nanorod arrays can be selectively grown on the gate area to immobilize glucose oxidase (GOx). The nanorods can be, for example, metal oxide and nitride based nanorods. Nanorod metal oxides include, but are not limited to, SnO, TiO ₂ , GaN, MgO, ZnMgO, and In ₂ O ₃ nanorods. For example, one-dimensional

ZnO nanorods on the gate area result in a very high specific surface area with high

surface to volume ratio and provide favorable micro-environments for the immobilization of GOx.

The AlGaN/GaN HEMT drain-source current has a rapid response, of less than 5 seconds, when glucose in a buffer with a pH value of 7.4 was added to the GOx immobilized ZnO nanorods surface. A wide range of glucose concentrations from to

0.5 nM to 125 μM can be detected. For example one sensor according to an embodiment of the invention exhibited a linear range from 0.5 nM to 14.5 μM with a limit of detection of 0.5 nM.

Following are examples that illustrate embodiments of the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 — Selective Detection of Hg(II) ions from Cu(II) and Pb(II)

Hg ² ' and Cu ²ⁱ ions are easily detected with sensors fabricated with Au-gated and thioglycolic acid functionalized Au-gated GaN/ AlGaN HEMTs.

The HEMT structures consisted of a 2 μm thick undoped GaN buffer and 250 A thick undoped Alo _.2 sGao ₇₅ N cap layer. The epi-layers were grown by molecular beam epitaxy system on 2" sapphire substrates at SVT Associates. Mesa isolation was performed with an Inductively Coupled Plasma (ICP) etching with CVAr based discharges at -90 V dc self-bias, ICP power of 300 W at 2 MHz and a process pressure of 5 mTorr. Ohmic contacts of 50 x 50 μm separated with gaps of 10, 20, and 50 μm were formed by e-beam deposition of Ti/Al/Pt/Au patterns by lift-off and annealed at 850 ⁰ C, 45 sec under flowing N ₂ for source and drain metal contacts. A 5- nm thin gold film was deposited as gate metal for two set of sample sensors. One sensor had a bare Au-gate and the other sensor had an Au-gate that was functionalized with a self-assembled monolayer of thioglycolic acid. An increase in the hydrophilicity of the surface treated with thioglycolic acid functionalization was confirmed by contact angle measurements which showed a change in contact angle from 58.4° to 16.2° after the surface treatment, as shown in FIGS. 5A, 5B. A 500- nm-thick poly(methyl methacrylate) (PMMA) film was used to encapsulate the source/drain regions, with only the gate region exposed to allow the liquid solutions to access the bare Au-gate or functionalized Au-gate surface. The source-drain current-

voltage characteristics were measured at 25 ⁰ C using an Agilent 4156C parameter analyzer with the Au-gated region exposed to different concentrations of Hg ^-* , Cu ²⁺ , Pb ^" , Mg ^" or Na solutions. AC measurements were performed to prevent side electrochemical reactions with modulated 500-mV bias at 11 Hz. A schematic cross-section of the device with Hg ^2' ions bound to thioglycolic acid functionalized on the gold gate region is shown in Figure 2. A self assembled monolayer of thioglycolic acid molecule was adsorbed onto the Au-gate due to strong interaction between gold and the thiol-group for the functionalized sensors. Excess thioglycolic acid molecules were rinsed from the monolayer using Dl water. XPS and electrical measurements confirmed a high surface coverage of thioglycolic acid molecules with Au-S bonding formation on the GaN surface.

Figure 6 shows the change in drain current of a bare Au-gated AlGaN/GaN HEMT sensor and a thioglycolic acid functionalized AlGaN/GaN HEMT sensor exposed to 10 ^~5 M Hg ²⁺ ion solutions as compared to exposed to DI water. The drain current of both sensors decreased after exposure to Hg ²⁺ ion solutions. The drain current reduction of the thioglycolic acid functionalized AlGaN/GaN HEMT sensors exceeded that of the bare Au-gate sensor by almost 80%. Though not to be bound by theory, the mechanisms of the drain current reduction for bare Au-gate and thioglycolic acid functionalized AlGaN/GaN HEMT sensors are probably quite different. For the thioglycolic acid fictionalized AlGaN/GaN HEMT, the thioglycolic acid molecules on the Au surface align with carboxylic acid functional group extending toward the solution. The carboxylic acid functional group of the adjacent thioglycolic acid molecules can form chelates (R-COO ^" (Hg ²⁺ )OOC-R) with the Hg ²⁺ ions. Upon chelation, one would expect the charges of trapped Hg ²⁺ ion in the R- CO(X(Hg ^{^+} )OOC-R to change the polarity of the thioglycolic acid molecules.

Because Hg ⁺ ions were used in our experiments, no Au-mercury amalgam is expected to form on the bare Au-surface.

Figure 7 shows time dependence of the drain current for the two types of sensors for detecting Hg ^" , Cu" , and Pb ^" ions. Both type of sensors showed very short response time (less than 5 seconds), when exposed to Hg ⁺ ion solution. The limits of detection for Hg" ⁺ ion detection for the bare Au-gate and thioglycolic acid functionalized sensor were 10 ^"6 and 10 ^"7 M, respectively. Neither sensor could detect

Pb ²⁺ ions. For the Cu ²⁺ ions, the detection limit of the thioglycolic acid functionalized sensor was around 10 ^"7 M, while the bare Au-gate could not detect the Cu ²⁺ ions as shown in Figure 7.

Figure 8 shows the drain current changes in response to Hg ^~* and CiT ⁺ ions as a function of the ion concentration for the two different surfaces. The difference in the response between the bare Au-gate and the thioglycolic acid functionalized sensor offers the possibility for selective detection for Hg ^"+ and Cu ^~+ ions presented in a single solution with a sensor chip containing both type of sensors, as shown in Figure

9. The dimension of the active area of the AlGaN/GaN HEMT sensor is less than 50 μm x 50 μm, and the sensors can be fabricated as an array of individual sensors. The fabrication of both sensors is identical except for the thioglycolic acid functionalized sensor, which has an additional functionalization step. This step can be accomplished with micro-inkjet system to locally functionalize surfaces. The bare Au-gate and thioglycolic acid functionalized sensors also showed excellent sensing selectivity (over 100 times higher selectivity) over Na ⁺ and Mg ²⁺ ions. As illustrated in Figure

10, there was almost no detection of Na ⁺ and Mg" ⁺ ions for both types of sensors with 0.1 M concentrations.

Most semiconductor based chemical sensors are not reusable. The bare Au- gate and thioglycolic acid functionalized sensors showed very good reusability, as shown in Figure 11. After a simple rinse with DI water, the sensors can be reused for

Hg ²⁺ ion detection repeatedly and the responses to different ionic solutions remain unchanged. The stability of thioglycolic acid functionalized Au surface is affected by several factors, like oxygen level, light, and initial packing quality. The subject devices were stored in nitrogen ambient and repeatedly used over a couple of weeks without substantial diminishment of efficacy.

The Hg ²⁺ /Ca ²⁺ sensor can operate at 0.5 V of drain voltage and 2 mA of drain current. However, the operation voltage and device size can be further reduced to minimize the power consumption to μW range. The sensor can be integrated with a commercial available hand-held wireless transmitter to realize a portable, fast response and high sensitivity Hg" ^"+ and Cu ion detector.

In summary, bared Au-gate and thioglycolic acid functionalized AlGaN/GaN HEMT sensors have demonstrable ability to detect heavy ions. The bare Au-gate

sensor was sensitive to Hg ^{2 f} and thioglycolic acid functionalized sensors could detect both Hg ^~+ and CiT ⁺ ions. By fabricating an array of the sensors on a single chip and selectively functionalizing some sensors with thioglycolic acid, a multi-functional specific detector can be fabricated. Such a sensor array can be used to detect quantitatively Hg" ^h ions in Cu ^" " ^f ion solution or CtT ions in Hg' ^' ion solution. Both bare Au-gate and thioglycolic acid functionalized sensor can be repeatedly used after a simple DI water rinse.

Example 2 — Detection of Prostate Specific Antigen Functionalization of Au-gated AlGaN/GaN high HEMTs of the invention were used to detect prostate specific antigen (PSA). The PSA was specifically recognized through PSA antibody, anchored to the gate area in the form of carboxylate succinimdyl ester. A wide range of concentrations from to 1 μg/ml to 10 pg/ml of PSA was investigated, which is lower than the cut-off \ alue of 2.5 ng/ml that is used as an indication for the need of biopsy.

The HEMT structures consisted of a 3 μm thick undoped GaN buffer, a 3θA thick AIo 3Gao ₇ N spacer, and a 22θA thick Si-doped Al _{0 3} Ga _{0 7} N cap layer. Epi-layers were grown by rf plasma-assisted Molecular Beam Epitaxy on the thick GaN buffers produced on sapphire substrates by metal organic chemical vapor deposition (MOCVD). Mesa isolation was performed with an Inductively Coupled Plasma (ICP) etching with Cl ₂ ZAr based discharges at -90 V dc self-bias, ICP power of 300 W at 2 MHz and a process pressure of 5 mTorr. 10 x 50 μm ² Ohmic contacts separated with gaps of 5 μm consisted of e-beam deposited Ti/Al/Pt/Au patterned by lift-off and annealed at 850 ⁰ C, 45 sec under flowing N ₂ . Poly(m ethyl methacrylate) (PMMA) was used to form 400-nm-thick 4% encapsulate the source/drain regions, with only the gate region exposed to allow the liquid solutions to contact the gate surface. The source-drain current-voltage characteristics were measured at 25°C using an Agilent 4156C parameter analyzer with the gate region exposed to solution. Ac measurements were performed with modulated 500-mV bias at 11 Hz to prevent side electrochemical reactions.

A plan view photomicrograph of a completed device and schematic cross- section of the device is shown in Figure 12. The Au surface was functionalized with a

specific bifunctional molecule. The thioglycolic acid, HSCH ₂ COOH, was used attached to the Au surface in the gate area, as a self assembled monolayer adsorbed on the gold gate.

The devices were first placed in the ozone/UV chamber for 3 minutes and then submerged in a 1 mM aqueous solution of thioglycolic acid for 24 hours at room temperature, resulting in binding of the thioglycolic acid to the Au surface in the gate area with the COOH groups available for further chemical functionalization. XPS and electrical measurements were taken to confirm a high surface coverage and Au-S bonding formation on the surface. The device was freshly cleaned with deionized water to remove unlinked thioglycolic acids. The carboxylic acid functional groups were activated by submerging the device in a 0.1 mM solution of N, N'- dicyclohexylcarbodiimide (DCC) in dry acetonitrile for 30 minutes and then in a 0.1 mM solution of N-hydroxysuccinimide in dry acetonitrile for 1 hour. These functionalization steps resulted in the formation of succinimidyl ester groups on the gate area of AlGaN/GaN HEMT, as shown in Figure 12B. The device was incubated in a phosphate buffered saline (PBS) solution of anti-PSA monoclonal antibody for 18 hours before real time measurement of PSA.

After incubation in a PBS buffered solution containing PSA at a concentration of 1 μg/ml, the device surface was thoroughly rinsed with deionized water and dried with a nitrogen stream. The electrical properties of the devices, source and drain current, were measured before and after PSA incubation as shown in Figure 13 A. The electrons in the two-dimensional electron gas (2DEG) channel of the AlGaN/GaN HEMT are induced by piezoelectric and spontaneous polarization effects. This 2DEG is located at the interface between the GaN layer and AlGaN layer. There are positive counter charges at the AlGaN surface layer induced by the 2DEG. Any slight changes in the ambient of the AlGaN/GaN HEMT affect the surface charges of the AlGaN/GaN HEMT. These changes in the surface charge are transduced into a change in the concentration of the 2DEG in the AlGaN/GaN HEMTs, leading to the slight decrease in the conductance for the device after PSA incubation. Figure 13B shows the real time PSA detection in PBS buffer solution using the source and drain current change with a constant bias of 500 mV. No current change can be seen with additional buffer solution around 100 sec and nonspecific

bovine serum albumin (BSA) around 200 sec, showing relatively high stability of the device and chemical surface modification. In clear contrast, the current change showed a rapid response of less than 5 seconds when target 10 ng/ml PSA was introduced to the antibody on the surface. The abrupt current change, mainly due to the exposure of PSA in a buffer solution, stabilized after the PSA diffused into buffer solution thoroughly.

Further real time test for the detection limit of PSA of less dilute concentrations was carried out as shown in Figure 14 A. Three different concentrations of the exposed target PSA in a buffer solution were observed from 10 pg/ml to 1 ng/ml. The amplitude of current change for the device exposed to PSA in a buffer solution was about 3%, as illustrated in Figure 14B. The clear current decrease of 64 nA as 10 pg/ml of PSA also indicated that the detection limit could be lowered up to several pg/ml, showing the promise of portable electronic biological sensor for PSA screening. As demonstrated herein, through a chemical modification sequence, the Au- gated region of an AlGaN/GaN HEMT structure can be functionalized for the detection of PSA with a sensitivity of 10 pg/ml in a buffer at clinical concentration. This electronic detection of biomolecules can occur with a compact sensor chip, which can be integrated with a commercial available hand-held wireless transmitter to realize a portable, fast and high sensitivity prostate cancer detector.

Example 3 — Detection of Changes in pH in Electrolyte Solutions

A Sc ₂ O ₃ gate dielectric on AlGaN/GaN HEMTs are shown to provide high sensitivity for detecting changes in pH of electrolyte solutions and is superior to the use of native oxide in the gate region.

The HEMT structures consisted of a 2 μm thick undoped GaN buffer and 25θA thick undoped Alo _.25 Gao _.75 N cap layer. The epi-layers were grown by Metal- Organic Chemical Vapor Deposition on 100 mm (111) Si substrates at Nitronex Corporation. The sheet carrier concentration was ~lxlθ ¹³ cm ^"2 with a mobility of 980 cm ² /V~s at room temperature. Mesa isolation was achieved by using an inductive coupled plasma system with Ar/Cl ₂ based discharges. Ohmic contacts of 50 x 50 μm ² separated with gaps of 10, 20, and 50 μm formed by lift-off of e-beam deposited

Ti(2OθA) /Al(800A) /Pt(4OθA) /Au(8OθA). The contacts were annealed at 850 ⁰ C for 45 sec under a flowing N ₂ ambient in a Heatpulse 610T system. A IOOA Sc ₂ O ₃ layer was deposited as a gate dielectric through a contact window of SiN _x layer. Before oxide deposition, the wafer was exposed to ozone for 25 minutes, and heated in-situ at 300 ⁰ C for 10 minutes inside the growth chamber. The Sc ₂ O ₃ was deposited by rf plasma-activated MBE at 100 ⁰ C using elemental Sc evaporated from a standard effusion all at 1130 ⁰ C and O ₂ derived from an Oxford RF plasma source.

For comparison, devices with just the native oxide present in the gate region and also with the UV ozone -induced oxide were fabricated. Figure 3 shows a scanning electron microscopy (SEM) image (top) and a cross-sectional schematic

(bottom) of the completed device. The gate dimension of the device is 2 x 150 μm . The pH solution was applied using a syringe autopipette (2-20 μl).

Prior to the pH measurements, pH 4, 7, 10 buffer solutions from Fisher Scientific were used to calibrate the electrode and the measurements at 25 °C were carried out in the dark using an Agilent 4156C parameter analyzer to avoid parasitic effects. The pH solution made by the titration method using HNO ₃ , NaOH and distilled water. The electrode was a conventional Acumet standard Ag/AgCl electrode.

The adsorption of aqueous solution of different pH on the surface of the HEMT affected the surface potential and device characteristics. Figure 15 shows the current at a bias of 0.25V as a function of time from HEMTs with Sc ₂ O ₃ in the gate region exposed for 150s to a series of solutions whose pH was varied from 3-10. The current significantly increased as the pH is decreased upon exposure to these aqueous solutions. The change in current was 37 μA/pH. The HEMTs show stable operation with a resolution of -0.1 pH over the entire pH range, showing the remarkable sensitivity of the HEMT to relatively small changes in concentration of the hydronium ion in solution. By comparison, devices with the native oxide in the gate region showed a higher sensitivity of ~70 μA/pA but a poor resolution of ~0.4 pH and showed delays in response of 10-15 seconds. The delays may result from deep traps at the interface between the semiconductor and native oxide, whose density is much higher than at the Sc ₂ O ₃ -nitride interface. The devices with UV-ozone oxide in the gate region did not show these incubation times for detection of pH changes and

showed similar sensitivities of gate source current as the Sc ₂ O ₃ gate devices (~40 μA/pH) but displayed poorer resolution(~0.25 pH). Figure 15 shows that the HEMT sensor with Sc ₂ O ₃ gate dielectric is sensitive to the concentration of the polar liquid and therefore could be used to differentiate between liquids into which a small amount of leakage of another substance has occurred.

The pH range of interest for human blood is 7-8. Figure 16 shows the current change in the HEMTs with Sc ₂ O ₃ at a bias of 0.25V for different pH values in this range. The resolution of the measurement is <0.1 pH. The electrons in the two- dimensional electron gas (2DEG) channel of the AlGaN/GaN HEMT are induced by piezoelectric and spontaneous polarization effects. This 2DEG is located at the interface between the GaN layer and AlGaN layer. There are positive counter charges at the AlGaN surface layer induced by the 2DEG. Any change in the ambient of the AlGaN/GaN HEMT affects the surface charges of the device. These changes in the surface charge are transduced into a change in the concentration of the 2DEG. Different pH exhibit different degrees of interaction with the ZnO surface. These results show that using a higher quality oxide is useful in improving pH resolution.

Example 4 — Detection of Kidney Injury Molecule- 1

A HEMT structure consisted of a 2 μm thick undoped GaN buffer and 25θA thick undoped Alo _.25 Gao _.75 N cap layer. The epi-layers were grown by metal-organic chemical vapor deposition on 100 mm (1 11) Si substrates. Mesa isolation was performed with Inductively Coupled Plasma (ICP) etching with Cl ₂ /Ar based discharges at -90 V dc self-bias, ICP power of 300 W at 2 MHz and a process pressure of 5 mTorr. Ohmic contacts of 50 x 50 μnT separated with gaps of 20 μm resulted from e-beam deposited Ti/Al/Pt/Au patterned by lift-off and annealed at 850

⁰ C for 45 sec under a N ₂ stream. A 5-nm thin gold film was deposited as a gate metal and functionalized as a self-assembled monolayer of thioglycolic acid. poly(methyl methacrylate) (PMMA) was used to form a 500-nm-thick encapsulate of the source/drain regions, with the gate region exposed using e-beam lithography. A plan view photomicrograph of a completed device is shown in Figure 17 A.

Before depositing the thioglycolic acid coating, the sample was exposed to UV ozone for 5 minutes to remove surface contamination. A self-assembled monolayer

of thioglycolic acid molecule was adsorbed onto the Au-gate. Excess thioglycolic acid was rinsed off with PBS buffer. An increase in the hydrophilicity of the treated thioglycolic acid functionalization surface was confirmed by contact angle measurements, which showed a change in contact angle from 58.4° to 16.2° after the surface treatment.

The thioglycolic acid surface was treated with monoclonal anti rat kidney injury molecule- 1 (KIM-I) antibody in a solution of 10 niM phosphate buffer containing 4 mM sodium cyano-borohydride, pH 8.8 at room temperature for 2 hours. This antibody immobilization is based on a strong reaction between carboxyl group on thioglycolic acid and amine group on KIM-I antibody. Excess KIM-I antibodies were washed from the surface using a PBS buffer and the unreacted surface carboxyl groups were passivated by a blocking solution of 100 mM ethanolamine in 10 mM phosphate buffer pH 8.8. Figure 17B shows a schematic device cross section with thioglycolic acid followed by KIM-I antibody coating. The source-drain current- voltage characteristics were measured at 25°C using an Agilent 4156C parameter analyzer with the KIM-I antibody functionalized Au-gated region exposed to different concentrations of KIM-I /PB S buffer. AC measurements were performed to prevent side electrochemical reactions with modulated 500-mV bias at 11 Hz.

The source and drain current(Ios) vs. voltage(Vos) for the devices, were measured in PBS buffer and 100 ng/ml KIM-I in PBS buffer, as shown in Figure 18.

There is a clear conductance decrease with KIM-I exposure and this suggests that through the selective binding of KIM-I with antibody, there are charges accumulated at the surface and these surface charges are transduced into a change in the carrier concentration of AlGaN/GaN 2DEG, leading to the obvious decrease in the conductance of the device after KIM-I exposure.

Figure 19 shows the time dependent source-drain current signal with a constant bias of 500 mV for KIM-I detection in PBS buffer solution. No current change can be seen with the addition of buffer solution around 50 sec. This stability excludes the possibility of noise due to the mechanical change of the buffer solution. By sharp contrast, the current change showed a rapid response in less than 20 seconds when target 1 ng/ml KIM-I was switched to the surface at 150sec. The abrupt current change due to the exposure of KIM-I in a buffer solution stabilized after the KIM-I

thoroughly mixed with the buffer. A lO ng/ml KIM-I solution was then applied at 350sec, which was accompanied by a larger signal due to the higher KIM-I concentration.

Additional real time tests were carried out to explore the limits of detection of KIM-I antibody (Figure 20). The device was exposed to 10 pg/ml, 100 pg/ml, 1 ng/ml,

10ng/ml, and 100ng/ml individually with each concentration repeated five times to determine the standard deviation of source-drain current response for each concentration. The limit of detection of this device was Ing/ml KIM-I in PBS buffer solution and the source-drain current change is nonlinearly proportional to the KIM-I concentration. Between each test, the device was rinsed with a wash buffer of 10 μM phosphate buffer solution containing 10 μM KCl with pH 6 to strip the antibody from the antigen. These results suggest that our HEMTs are compatible with AKI biomarker, KIM-I, are very sensitive relative to other currently available nano- devices, and are useful for preclinical and clinical applications. Similar surface modifications can be applied for detecting other important disease biomarkers and a compact disease diagnosis array can be realized for multiplex disease analysis. Example 5 — Glucose Detection

A HEMT structure consisting of a 3 μm thick undoped GaN buffer, 3θA thick Alo _.3 Gao. ₇ N spacer, 220 A thick Si-doped Alo _.3 Gao _.7 N cap layer was provided. Epi-layers were grown by both molecular beam epitaxy and metal organic chemical vapor deposition (MOCVD) on thick GaN buffers on sapphire substrates. Mesa isolation was performed with an Inductively Coupled Plasma (ICP) etching with Cl ₂ / Ar based discharges at -90 V dc self-bias, ICP power of 300 W at 2 MHz and a process pressure of 5 mTorr. 50 x 50 μm ² Ohmic contacts separated with gaps of 10 μm consisted of e-beam deposited Ti/Al/Pt/Au patterned by lift-off and annealed at

850 ⁰ C, 45 sec under flowing N ₂ .

ZnO nanorods were grown in a solution of 20 mM zinc acetate hexahydrate (Zn(NO ₃ ) ₂ -6H ₂ O) and 20 mM hexamethylenetriamine (C ₆ Hi ₂ N ₄ ) in a flask with polypropylene autoclavable cap at a controlled temperature and pH on the HEMT substrate. Subsequently, the substrate was removed from solution, thoroughly rinsed with acetone followed by deionized water to remove any residual salts and dried in air at room temperature. The ZnO nanoparticle size was highly dependent on the

nanocrystal seed preparation time. The diameters of ZnO nanorods can be carefully controlled from tens of nanometers to several hundred micrometers by the seed size used to grow the nanorods. Figure 21 shows the effect of nanocrystal seed size on the diameters of ZnO nanorods grown on a Si (111) substrate. By incorporating the nanorods on the HEMT gate sensing area, the total sensing area increases significantly as shown in Figure 22a. The conventional AlGaN/GaN HEMT detects the ambient changes through the "gate sensing area". This area is defined as gate length x gate width in the regular HEMT. Although, we can increase the gate width to gain higher drain current from the transistor, the sensor detection sensitivity will be the same for HEMT with both short and longer gate width. This is due to the signal and background current proportionally increasing at the same time. The other dimension of the gate is the gate length. However, increasing the gate length increases the parasitic resistance of the HEMT and the drain current decreases. Thus, the detection sensitivity goes down. Therefore, the only way to increase the sensitivity with the same "gate dimension" is to grow 3D structures on the gate sensing area to increase the total sensing area with the area expansion to the third dimension.

Figure 22b show SEM pictures of ZnO nanorods grown on the AlGaN/GaN HEMT gate sensing area. The upper right inset in Figure 22 shows a closer view of ZnO nanorod arrays grown on the gate area with different scales. The ZnO nanorods matrix provides a microenvironment for immobilizing negatively charged GOx and retains its bioactivity, and passes charges produce during the GOx and glucose interaction to the AlGaN/GaN HEMT.

A GOx solution was prepared with a concentration of 10 mg/mL in the 10 mM phosphate buffer saline (pH value of 7.4., Sigma Aldrich). After fabricating the device, 5 μl GOx (~100 units/mg, Sigma Aldrich) solution was dropped on the surface of HEMT device. The HEMT device was kept at 4 ⁰ C in the solution for 48 hours for GOx immobilization on the ZnO nanorod arrays followed by an extensive washing to remove the unimmobilized GOx. The HEMT device was kept in the incubator for 30 minutes to make the enzyme active around 37 ⁰ C.

The target glucose was applied on the device through a syringe autopipette (2- 20μl). The current-voltage characteristics were measured using an Agilent 4156C parameter analyzer with the gate region exposed.

Figure 23 shows the real time glucose detection in PBS buffer solution using the drain current change with constant bias of 250 mV. No current change can be seen with the addition of buffer solution at around 200 sec, showing the specificity and stability of the device. By sharp contrast, the current change showed a rapid response in less than 5 seconds when target glucose was added to the surface. The response is very linear from 0.5 nM to 14.5 μM and an experimental limit of detection of 0.5 nM.

Immobilization of the negatively charged GOx on the positively charged ZnO nanorod arrays is maximized around the pH value of 7.4 and reduces to about 80% for pH - 5 to 6. Once the pH value is larger than 8, the activity drops significantly. The human pH value can vary depending on the health condition, e.g. the pH value for patients with acute asthma was reported as low as 5.23 + 0.21 (n=22) as compared to

7.65 + 0.20 (n=19) for the control subjects. In order to get accurate measurements of glucose concentration in the EBC, one needs to know the correlation of the pH value of the EBC and the sensitivity of GOx functionalized for specific sensors. The low detection limit of the sensor permitted dilution of < 0.1 μ-liter of exhaled breath condensate (EBC) in 100-200 μ-liter PBS and the direct measurement of the glucose concentration to eliminate the effect of pH variation of the EBC. Figure 24 shows the changes of drain current as a function of glucose concentration. A very good linear relationship of glucose concentration vs. drain current changes was obtained. Because of the fast response time and low volume of the EBC required, a handheld real-time glucose sensor can be made.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all Figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light

thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.