sensors Article

A Micro-Platinum Wire Biosensor for Fast and Selective Detection of Alanine Aminotransferase Tran Nguyen Thanh Thuy and Tina T.-C. Tseng * Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan; [email protected] * Correspondence: [email protected]; Tel.: +886-2-273-766-52 Academic Editor: Roberto Pilloton Received: 21 April 2016; Accepted: 23 May 2016; Published: 26 May 2016

Abstract: In this study, a miniaturized biosensor based on permselective polymer layers (overoxidized polypyrrole (Ppy) and Nafion® ) modified and enzyme (glutamate oxidase (GlutOx)) immobilized micro-platinum wire electrode for the detection of alanine aminotransferase (ALT) was fabricated. The proposed ALT biosensor was measured electrochemically by constant potential amperometry at +0.7 V vs. Ag/AgCl. The ALT biosensor provides fast response time (~5 s) and superior selectivity towards ALT against both negatively and positively charged species (e.g., ascorbic acid (AA) and dopamine (DA), respectively). The detection range of the ALT biosensor is found to be 10–900 U/L which covers the range of normal ALT levels presented in the serum and the detection limit and sensitivity are found to be 8.48 U/L and 0.059 nA/(U/L¨ mm2 ) (N = 10), respectively. We also found that one-day storage of the ALT biosensor at ´20 ˝ C right after the sensor being fabricated can enhance the sensor sensitivity (1.74 times higher than that of the sensor stored at 4 ˝ C). The ALT biosensor is stable after eight weeks of storage at ´20 ˝ C. The sensor was tested in spiked ALT samples (ALT activities: 20, 200, 400, and 900 U/L) and reasonable recoveries (70%~107%) were obtained. Keywords: alanine aminotransferase; microelectrode; amperometric; glutamate oxidase; biosensor

1. Introduction Liver diseases can often lead to failing of normal liver functions (e.g., metabolic function, digestion, protein synthesis, detoxification, etc.). The development of advanced biosensing techniques plays an important role in the diagnosis of liver diseases and tracing liver conditions and therefore, these diseases may be controlled or treated if identified early. Common liver function tests often contain measurements of bilirubin, albumin, transferases (e.g., alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), etc.), and other enzymes (e.g., alkaline phosphatase (AP), lactate dehydrogenase (LDH), etc.) [1]. Among them, aminotransferases (ALT and AST) are the most useful measures of hepatic injury. On the other hand, when hepatocytes are damaged, the ALT level can increase up to 50 times greater than normal; therefore, compared to AST, the ALT level is more liver specific [2]. ALT can be found in cardiac muscles, skeletal muscles, kidneys, and mainly in the liver. The normal ALT concentration in the blood is ~5–35 U/L (the upper limit threshold of ALT level is generally ~40 U/L) [2,3]. Abnormalities of ALT activity in the serum may be associated with some chronic liver diseases such as alcoholic liver disease, chronic hepatitis B or C, hepatocellular carcinoma, etc. [1] A variety of analytical approaches have been used to determine the ALT level in liver function tests, such as colorimetry [4–6], spectrophotometry [7,8], chemiluminescence [9], chromatography [10], fluorescence [11–13], electrochemical techniques [14–18], etc. When techniques like fluorescence, chromatography, or chemiluminescence are used, careful conductions of tests are required since tests could be affected by environmental factors (e.g., pH, light intensity, temperature, ionic strength, Sensors 2016, 16, 767; doi:10.3390/s16060767

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interactions in solutions, etc.) easily [19] and therefore result in low sensitivity for measuring ALT [2]. Spectrophotometry is the most routinely used method in the clinical laboratory which can provide precise and accurate detection of ALT; however, performing spectrophotometric measurements usually consumes large quantities of expensive reagents and requires cumbersome instruments and time-consuming incubation steps [3,20]. Recently, increased attention has been drawn to electrochemical techniques, including differential pulse voltammetry [21], chronoamperometry [22], cyclic voltammetry [23], constant potential amperometry [3,14,16–18,24–29], etc., for the detection of ALT; in general, they provide fast response time and high sensitivity for measuring ALT; besides, they require only compact instruments and straightforward operations. Different materials including gold (Au) [21,22], palladium (Pd) [28], graphite [23], platinum (Pt) [3,18,25–27], and other materials [14,17,24] for the fabrication of ALT biosensors were reported. Among them, Pt is more stable and inert [29,30] and shows better electrooxidation properties for hydrogen peroxide (H2 O2 ), the product of some enzymatic reactions catalyzed by oxidases in presence of water and oxygen; therefore, Pt is a superior electrode material for fabricating biosensors using oxidases as biorecognition elements. In general, electrodes with small dimensions are preferred since the cost of sensor fabrication as well as the volume of tested samples can be reduced. To increase the sensitivity and selectivity of ALT biosensors, different enzymes working as biorecognition elements have been used to modify the electrode surface, including pyruvate oxidase (PyOx) [14,16,24], lactate dehydrogenase (LDH) [21], glutamate oxidase (GlutOx) [3,17,18,25–28], etc. In fact, GlutOx-based ALT biosensors have much higher storage stability and simpler fabrication process; besides, they do not require cofactors during measurements, whereas PyOx-based ALT biosensors require the addition of thiamine pyrophosphate and Mg2+ for testing [17,18]. Different modification layers including cellulose acetate [18] and Nafion® [28] have been used during the fabrication of ALT biosensors to eliminate interference from the negatively charged ascorbic acid (AA) and uric acid (UA) presented in testing samples [18,23,27,28]; however, there are few study reporting on ALT biosensors which can reject both positively and negatively charged interferents. In this study, micro-Pt wire is chosen to be the base (i.e., the working electrode) for the fabrication of the ALT biosensor. The biocatalytic reaction involved in the process of measuring ALT on the ALT biosensor is based on the enzymatic reaction of the target ALT producing L-glutamate and pyruvate in presence of L-alanine and α-ketoglutarate (Equation (1)); further oxidation of L-glutamate by glutamate oxidase (GlutOx) incorporated on the ALT biosensor yields H2 O2 , α-ketoglutarate, and ammonia (Equation (2)). The overall working principle of the ALT biosensor that we proposed is shown in Figure 1. The surface of the Pt working electrode was modified with the overoxidized Ppy layer in order to exclude electroactive interferents, such as ascorbic acid (AA) and dopamine (DA), and then the Nafion® layer in order to reject anionic interferents [31,32] for enhancing the selectivity of the ALT biosensor. GlutOx was immobilized on the electrode atop those permselective modification layers for oxidizing L-glutamate produced from the previous enzymatic reaction (Equation (1)) and generating H2 O2 . The generated H2 O2 is a small and neutral molecule which can permeate through permselective layers and reach the electrode surface. The electrooxidation of H2 O2 occurs on the electrode surface when a constant potential (0.7 V vs. Ag/AgCl) applies (Equation (3)). The measured current signals can be correlated to ALT levels by calibration. In this work, a fast, small, and economic ALT biosensor is proposed for assisting the development of a convenient diagnosing technique for liver diseases. ALT

L ´ alanine ` α ´ ketoglutarate ÝÝÑ L ´ glutamate ` pyruvate GlutOx

(1)

L ´ glutamate ` O2 ÝÝÝÝÑ α ´ ketogularate ` NH3 ` H2 O2

(2)

H2 O2 Ñ O2 ` 2H` ` 2e´

(3)

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Figure1. 1. The The schematic schematic diagram diagramshowing showingthe theworking workingprinciple principleof ofthe theALT ALTbiosensor. biosensor. Figure

2.2. Materials Materials and and Methods Methods

2.1.Materials Materials 2.1. Pyrrole, L-alanine, L-alanine, α-ketoglutaric α-ketoglutaric acid acid sodium sodium salt, salt, and and 3-hydroxytyramine 3-hydroxytyramine hydrochloride hydrochloride Pyrrole, (dopamine hydrochloride, hydrochloride, DA) DA) were were purchased purchased from from ACROS ACROS Organics™ Organics™ (Bridgewater, (Bridgewater, NJ, NJ, USA). USA). (dopamine ® ® Nafion (perfluorinated ion solution in in lower aliphatic alcohols/H 2O mix) Nafion ion exchange exchangeresin, resin,5 5wt wt%%inin solution lower aliphatic alcohols/H 2O was purchased from Aldrich (Milwaukee, WI, USA). Bovine serum albumin ~66 kDa, purity 98%, mix) was purchased from Aldrich (Milwaukee, WI, USA). Bovine serum(BSA, albumin (BSA, ~66>kDa, lyophilized alanine aminotransferase (ALT, EC 2.6.1.2, from porcine purity > 98%,powder), lyophilized powder), alanine aminotransferase (ALT, EC 2.6.1.2, from heart, porcineactivity: heart, ≥75 U/mg protein), and glutaraldehyde (GAH) (GAH) were purchase from Sigma-Aldrich (St. Louis, MO, activity: ě75 U/mg protein), and glutaraldehyde were purchase from Sigma-Aldrich (St. Louis, USA). L-glutamate oxidase (GlutOx, ECEC 1.4.3.11) was from USA). MO, USA). L-glutamate oxidase (GlutOx, 1.4.3.11) was fromUS USBiological Biological(Marblehead, (Marblehead, MA, USA). ® (Farmingdale, ® (Farmingdale, L-(+)-ascorbicacid acid(AA) (AA)was was from Enzo USA). Hydrogen peroxide L-(+)-ascorbic from Enzo NY,NY, USA). Hydrogen peroxide (30% (30% w/v) w/v) was was from Panreac (Barcelona, Spain). other chemicals were analytical grade.Sodium Sodiumphosphate phosphate from Panreac (Barcelona, Spain). All All other chemicals were of of analytical grade. buffer(PBS, (PBS,pH pH composed 50 sodium mM sodium phosphate (dibasic) mMchloride. sodium buffer 7.4)7.4) waswas composed of 50ofmM phosphate (dibasic) and 100 and mM 100 sodium chloride. The perfluoroalkoxy (PFA) coated platinum wire (inner µm) used the for The perfluoroalkoxy (PFA) coated platinum wire (inner diameter: 50.8diameter: µm) used50.8 for preparing preparing the working electrodefrom was obtained from (Sequim, A-M Systems (Sequim, WA, USA). Ag/AgCl working electrode was obtained A-M Systems WA, USA). Ag/AgCl glass-bodied glass-bodied reference electrodes with 3 M NaCland electrolyte and platinum wire (diameter: 0.5 mm) reference electrodes with 3 M NaCl electrolyte platinum wire (diameter: 0.5 mm) auxiliary auxiliary electrodes were purchased Ltd. (Tokyo, Japan). wire (diameter: 203used µm) electrodes were purchased from ALSfrom Co.,ALS Ltd.Co., (Tokyo, Japan). The AgThe wireAg (diameter: 203 µm) used for preparing the Ag/AgCl wire reference electrode was obtained from RoHS (Taipei, Taiwan). for preparing the Ag/AgCl wire reference electrode was obtained from RoHS (Taipei, Taiwan). 2.2. 2.2.Instrumentation Instrumentation The The electrochemical electrochemical preparation preparation of of ALT ALTbiosensors biosensorsand andelectrochemical electrochemical measurements measurements were were conducted using a versatile multichannel potentiostat (model VSP 300, Bio-Logic SAS, Claix, France) conducted using a versatile multichannel potentiostat (model VSP 300, Bio-Logic SAS, Claix, France) in inaathree-electrode three-electrodeconfiguration configurationconsisting consistingof ofaaPt Ptwire wire(inner (innerdiameter: diameter: 50.8 50.8 µm) µm) working working electrode, electrode, aaPtPt wire (diameter: 0.5 mm) electrode, and a reference consisting of aconsisting glass-enclosed wire (diameter: 0.5 auxiliary mm) auxiliary electrode, and aelectrode reference electrode of a Ag/AgCl wire in 3 M NaCl solution or a Ag/AgCl wire (inner diameter: 203 µm), respectively. glass-enclosed Ag/AgCl wire in 3 M NaCl solution or a Ag/AgCl wire (inner diameter: 203 µm), All potentials are vs. are the Ag/AgCl reference electrode. The Ag/AgCl wire The reference electrode respectively. Allreported potentials reported vs. the Ag/AgCl reference electrode. Ag/AgCl wire was prepared by immersing the Ag wire (inner diameter: 203 µm) in 0.1 M HCl solution and then reference electrode was prepared by immersing the Ag wire (inner diameter: 203 µm) in 0.1 M HCl allowing reaction occur using a regular battery V) to form on the solution the andelectrolysis then allowing the to electrolysis reaction to AA occur using(1.5 a regular AAAgCl battery (1.5anode V) to (i.e., theAgCl Ag wire). Field emission scanning electron microscope JSM-7001F, Peabody, MA, form on the anode (i.e., the Ag wire). Field emission(model scanning electronJEOL, microscope (model USA) was used for magnifying the surface of ALT biosensors. JSM-7001F, JEOL, Peabody, MA, USA) was used for magnifying the surface of ALT biosensors. 2.3. 2.3.Preparation PreparationofofALT ALTBiosensors Biosensors The The Pt Ptwire wiremicroelectrode microelectrode was was prepared prepared by by stripping stripping two two ends ends of of aa piece piece of of 1.5 1.5 cm cm Pt Ptwire wire (inner diameter: 50.8 µm) covered by PFA to reveal 2.5 mm bare Pt wire on each end. Then, one (inner diameter: 50.8 µm) covered by PFA to reveal 2.5 mm bare Pt wire on each end. Then, oneend end (the connection end) was soldered with a piece of 8 cm long copper wire (AWG = 30) and the solder knot (the connection end) was soldered with a piece of 8 cm long copper wire (AWG = 30) and the solder was with the epoxy the other end of the exposed Pt will bePt used working knotcovered was covered with the glue; epoxy glue; the other end of the exposed willasbethe used as theelectrode working for the preparation of ALT biosensors. Once the epoxy glue was dried, the electrode was ready to use. electrode for the preparation of ALT biosensors. Once the epoxy glue was dried, the electrode was Right before use, the Pt working electrode was cleaned by sonicating with isopropyl alcohol. ready to use. Right before use, the Pt working electrode was cleaned by sonicating with isopropyl alcohol.

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For the of permselective polymer layers layers of of ALT ALT biosensors, the Ppy Ppy film film was For the construction construction of permselective polymer biosensors, the was electrodeposited on the surface of Pt wire working electrodes by cyclic voltammetry method (0.2–1.2 V, electrodeposited on the surface of Pt wire working electrodes by cyclic voltammetry method (0.2–1.2 V, 26 cycles, cycles,scan scanrate rate mV/s) 20pyrrole mM pyrrole in PBS. Then, these electrodes were 26 20 20 mV/s) in 20in mM solutionsolution in PBS. Then, these electrodes were dip-coated ® and baked at 180 ˝ C (3 min); the dip-coating and baking process was dip-coated with Nafion ® with Nafion and baked at 180 °C (3 min); the dip-coating and baking process was repeated two more repeated more The to Ppy filmcommon was usedpositively to reject common positively charged times. Thetwo Ppy filmtimes. was used reject charged interferents, such asinterferents, DA, while ® suchNafion as DA, whilewas the used Nafion layer common was usednegatively to reject common ® layer the to reject charged negatively interferents,charged such asinterferents, AA. These such as AA. These permselective polymer layerstowould contribute to increase thebiosensors selectivitytowards of ALT permselective polymer layers would contribute increase the selectivity of ALT biosensors the analyte and against interferents prevent the false positive current responses the analyte towards against interferents prevent the falseand positive current responses from interferents. fromThe interferents. enzyme layer was constructed atop permselective polymer layers by dip-coating. The Thesolution enzyme for layer was constructed atop permselective polymer by dip-coating. The the enzyme enzyme deposition was prepared by mixing the GlutOxlayers solution (250 U/mL) and BSA solution for deposition mixing the GlutOx solution (250 U/mL) theelectrode BSA solution solution (150 mM BSAwas andprepared 53 mM by GAH in DIW) in 1 to 1 volumetric ratio.and The was (150 mM BSA and 53 mM GAH in DIW) in 1 to 1 volumetric ratio. The electrode was dip-coated dip-coated with the enzyme solution for 90 times. The resulting ALT biosensors were stored at −20 °C ˝ C overnight with the enzyme solution for 90 times. The resulting ALT were stored at ´20and overnight prior to use. The cross-sectional structure ofbiosensors the bare Pt wire electrode the ALT prior to use. Thedemonstrated cross-sectional of the bare Pt thickness wire electrode ALTthe biosensor were biosensor were in structure Figure 2. Approximate of theand Ppythe layer, Nafion layer, demonstrated in Figure 2. Approximate thickness of the Ppy layer, the Nafion layer, and the enzyme and the enzyme layer were ~0.01 µm, ~0.6 µm, and ~0.1 µm estimated from the SEM picture. layer were ~0.01 µm, ~0.6 µm, and ~0.1 µm estimated from the SEM picture.

Figure 2. 2. The The cross-sectional cross-sectional structure structure of (b) the the ALT ALT biosensor biosensor Figure of (a) (a) the the bare bare Pt Pt wire wire electrode electrode (ˆ1000); (×1000); (b) (ˆ1500); and(c) (c)the theALT ALTbiosensor biosensor (ˆ20,000); layer Pt wire; (2): Overoxidased Ppy; (×1500); and (×20,000); layer (1): (1): BareBare Pt wire; layerlayer (2): Overoxidased Ppy; layer ® ; and layer (4): Immobilized enzyme layer. layer (3): Nafion ® (3): Nafion ; and layer (4): Immobilized enzyme layer.

2.4. Calibration Calibration of of ALT ALT Biosensors 2.4. Biosensors by by Method Method 11

The calibration measurements were conducted by constant potential amperometry at 0.7 V and corresponding to different ALT activities time were independently. the current currentresponses responses corresponding to different ALT vs. activities vs. recorded time were recorded In Method 1, the testing solution was a 2.5 mL stirring PBS with ALT substrates (100 mM L-alanine and independently. In Method 1, the testing solution was a 2.5 mL stirring PBS with ALT substrates 10 mM α-ketoglutaric Theα-ketoglutaric purchased ALT solid (1.61 mg, 124 ALT U/mg solid) was diluted with (100 mM L-alanine andacid). 10 mM acid). The purchased solid (1.61 mg, 124 U/mg 1solid) mL DIW to make 200 U/mL ALT stock solution; then, the stock solution was divided into 20 tubes was diluted with 1 mL DIW to make 200 U/mL ALT stock solution; then, the stock solution was ˝ C. Prior to use, the ALT stock solution was warmed up to the room (50 µL per tube) and stored atper ´20tube) divided into 20 tubes (50 µL and stored at −20 °C. Prior to use, the ALT stock solution was temperature and diluted with 150and µL diluted DIW to again make 50 U/mL ALTDIW solution. 2 µL,5010U/mL µL, 20ALT µL, warmed up to the room again temperature with 150 µL to make and 45 µL of the ALT solution (50 U/L) were injected into testing solutions directly and independently solution. 2 µL, 10 µL, 20 µL, and 45 µL of the ALT solution (50 U/L) were injected into testing to make solutions withindependently different ALT activities (40 U/L, with 200 U/L, 400 U/L, 900 U/L, respectively) solutions directly and to make solutions different ALT and activities (40 U/L, 200 U/L, and the current responsesand werethe recorded independently. Theresponses typical I-t were curve recorded obtained 400 U/L,corresponding and 900 U/L, respectively) corresponding current from testing ALT biosensors Method 1 was shown in Figure The slope obtained calculated independently. The typical I-tbycurve obtained from testing ALT3.biosensors by Methodwas 1 was shown within 603.s The (i.e.,slope fromobtained 30th s towas 90thcalculated s). The mean value of 030th U/L ALT determined by in Figure withinslope 60 s (i.e., from s to 90thwas s). The mean slope calculating theALT average background current before each injection of ALT. The calibration value of 0 U/L was stable determined by calculating the average stable background current beforecurve each was established plotting slopescurve of current activities. injection of ALT.byThe calibration was responses establishedvs.byALT plotting slopes of current responses vs. ALT activities.

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Figure 3. 3. The The typical typical I-t I-t curve curve when when testing testing ALT ALTbiosensors biosensors by byMethod Method 1. 1. The The injection injection timing timing of of the the Figure ALTsolution solutionisisat at20 20s.s. ALT

2.5. Calibration Calibration of of ALT ALT Biosensors Biosensorsby byMethod Method22 2.5. Thecalibration calibrationmeasurements measurementswere wereconducted conducted constant potential amperometry at V 0.7and V and The byby constant potential amperometry at 0.7 the the current responses corresponding to different ALT activities time were recorded plot. In current responses corresponding to different ALT activities vs. timevs. were recorded in a plot. in In aMethod 2, Method 2, the starting calibration solution was a 2 mL stirring PBS with ALT substrates (100 mM the starting calibration solution was a 2 mL stirring PBS with ALT substrates (100 mM L-alanine and L-alanine and 10 mM α-ketoglutaric acid).ALT The solution 50 U/L ALT was asin mentioned in 10 mM α-ketoglutaric acid). The 50 U/L wassolution prepared as prepared mentioned Section 2.4. Section 2.4.ALT To prepare ALTthe calibrators, the (50 ALTU/mL) solution (50mixed U/mL)with were mixed with substrate To prepare calibrators, ALT solution were substrate solutions (100 solutions (100 mM L-alanine and 10 mM α-ketoglutaric acid) to make eight ALT calibrators (ALT mM L-alanine and 10 mM α-ketoglutaric acid) to make eight ALT calibrators (ALT activities ranging activities fromU/mL, 0.21 U/mL, 0.23 U/mL, U/mL, U/mL, U/mL, U/mL, U/mL, from 0.21 ranging U/mL, 0.23 0.48 U/mL, 0.76 0.48 U/mL, 0.820.76 U/mL, 5.30.82 U/mL, 8.4 5.3 U/mL, to 8.4 9 U/mL). to 9 ALT-substrate U/mL). The ALT-substrate was prepared everylower 30 seconds (from lower to higher The mixture wasmixture prepared every 30 s (from to higher ALT activities) andALT let activities) and let each ALT-substrate mixture react for 10 minutes before injecting it into the each ALT-substrate mixture react for 10 min before injecting it into the testing solution (PBStesting with solution (PBS with mM α-ketoglutaric L-alanine andacid) 10 mM α-ketoglutaric acid) for the electrochemical 100 mM L-alanine and100 10 mM for the electrochemical measurement. The current measurement. The current response of the stirring testing solution (2 mL solution without was response of the stirring testing solution (2 mL solution without ALT) was first recorded forALT) 30 s and first recorded for 30 s and the current response of the blank was obtained. After the 10 min-reaction the current response of the blank was obtained. After the 10 min-reaction of the first ALT-substrate of the first ALT-substrate (calibrator 100injected µL of the calibrator was injected into the mL mixture (calibrator 1), 100mixture µL of the calibrator1),was into the 2 mL testing solution and2 the testing solution and the corresponding current response of 10 U/L ALT was recorded for another 30 s. corresponding current response of 10 U/L ALT was recorded for another 30 s. The same procedure The same procedure was followed for the electrochemical measurements of calibrator 2 to calibrator was followed for the electrochemical measurements of calibrator 2 to calibrator 8 and current responses8 and current responsesincreased of final ALT U/L, 40 U/L, 70 U/L, U/L, 300 U/L U/L, of final ALT activities fromactivities 20 U/L, increased 40 U/L, 70from U/L,20 100 U/L, 300 U/L, 600100 U/L, to 900 600 U/L, to 900 U/L were recorded. The calibration curve obtained by Method 2 was established by were recorded were recorded. The calibration curve obtained by Method 2 was established by plotting plotting current responses vs. ALT activities. current responses vs. ALT activities. 2.6. Determination Determination of of ALT ALT Activities Activities from fromSpiked SpikedSamples Samples 2.6. Spiked ALT samples were tested andand the measured ALT ALT activities were Spiked samples with withknown knownactivities activities were tested the measured activities compared to the actual known ALT activities. electrochemical were compared to the actual known ALT activities.The Thesetup setupand and recording recording of electrochemical measurements for for determining determining ALT ALT activities activities from from spiked spiked samples samples was was similar similar to to that that of of calibration calibration measurements measurements. In Method 1, current responses of spiked ALT ALT solutions solutions corresponding corresponding to to different different measurements. known ALT activities (20 U/L, 200 U/L, 400 U/L, and 900 U/L) were measured by the same procedure known ALT activities (20 U/L, 200 U/L, U/L, and 900 U/L) were measured procedure as mentioned mentionedin inSection Section2.4. 2.4.Based Basedon onthe thecalibration calibrationcurve curve obtained from calibration measurements as obtained from calibration measurements of of ALT biosensors tested by Method 1, activities of spiked samples (x) can be calculated. ALT biosensors tested by Method 1, activities of spiked ALTALT samples (x) can be calculated. In Method Method 2, 2, the the starting starting solution solution was was aa 3 mL stirring spiked ALT ALT samples samples in in PBS PBS with with substrates substrates In (100 mM mM L-alanine L-alanine and and 10 10 mM mM α-ketoglutaric α-ketoglutaric acid). acid). In this this experiment, experiment, four individual spiked ALT ALT (100 samples with different ALT activities (20 U/L, and 900900 U/L) were prepared. To samples U/L,200 200U/L, U/L,400 400U/L, U/L, and U/L) were prepared. prepare these spiked ALT samples, the ALT stock solution was mixed with the substrate solution To prepare these spiked ALT samples, the ALT stock solution was mixed solution (100 mM L-alanine α-ketoglutaric acid) for allowing ALT ALT to react for 10 min. (100 L-alanineand and1010mM mM α-ketoglutaric acid) for allowing to with reactsubstrates with substrates for One minute before the reaction ended, the first ALT calibration mixture (6.1 U/mL) was prepared in 10 min. One minute before the reaction ended, the first ALT calibration mixture (6.1 U/mL) was PBS with substrates and the other three ALT calibration mixtures (6.3 U/mL, 6.5 U/mL, and 6.7 U/mL) were prepared every 30 s sequentially. Once the spiked ALT sample had reacted for 10 min, its

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prepared in PBS with substrates and the other three ALT calibration mixtures (6.3 U/mL, 6.5 U/mL, corresponding responseevery (y1 in30 Figure 4) was recorded for 30 s. Subsequently, mL of substrate and 6.7 U/mL) current were prepared s sequentially. Once the spiked ALT sample3 had reacted for solution (without ALT) was injected into the testing solution and the current response of the diluted 10 min, its corresponding current response (y1 in Figure 4) was recorded for 30 s. Subsequently, 3 mL of testing solution 2 in Figure 4) was recorded for another 30 s. Afterwards, 100 µL the first ALT substrate solution(y(without ALT) was injected into the testing solution and the current response of the calibration mixture was (y added to the testing solution and its corresponding current response (y3 in diluted testing solution 2 in Figure 4) was recorded for another 30 s. Afterwards, 100 µL the first Figure 4) was recorded for 30 s. At moment, the ALT in the testing solution was ALT calibration mixture was added to this the testing solution andactivity its corresponding current response (100 + 0.5x) U/L where x is the activity of the spiked ALT sample. The similar procedure was repeated (y3 in Figure 4) was recorded for 30 s. At this moment, the ALT activity in the testing solution was for the addition the second the forth calibration mixture and The current responses (y4~ywas 6 in Figure 4) (100 + 0.5x) U/L of where x is theto activity of the spiked ALT sample. similar procedure repeated corresponding to (200 + 0.5x) U/L, (300 + 0.5x) U/L, and (400 + 0.5x) U/L were recorded. the I-t for the addition of the second to the forth calibration mixture and current responses (y4 ~y6 Once in Figure 4) curve (Figure 4) was obtained, the slope (a) of the linear calibration equation acquired from the corresponding to (200 + 0.5x) U/L, (300 + 0.5x) U/L, and (400 + 0.5x) U/L were recorded. Once the calibration data (y 6 vs. (100 + 0.5x) U/L~ (400 + 0.5x) U/L) could be calculated. This slope a was I-t curve (Figure 4)3~y was obtained, the slope (a) of the linear calibration equation acquired from the plugged into to Equations (4) and (5). After and be (5)calculated. simultaneously, the activity calibration data (y3 ~y6 vs. (100 + 0.5x) U/L~solving (400 + Equations 0.5x) U/L)(4) could This slope a was of the spiked sample could(4) beand determined on Equations our ALT biosensors by Method 2. The same plugged into to Equations (5). Afterbased solving (4) and (5)tested simultaneously, the activity procedure was repeated for determining the activities of other spiked ALT samples (200 U/L, 400 U/L, of the spiked sample could be determined based on our ALT biosensors tested by Method 2. The same and 900 U/L). procedure was repeated for determining the activities of other spiked ALT samples (200 U/L, 400 U/L, and 900 U/L).

= + y1 “ ax ` b x ax + = y2 “ 2 ` b 2

(4) (4) (5) (5)

Figure Thetypical typicalI-tI-tcurve curvewhen when testing ALT biosensors in spiked samples by Method 2. Figure 4. 4. The testing ALT biosensors in spiked samples by Method 2. The The measured current signals were represented by y ~y . 4 6 measured current signals were represented by y4~y6.

3. Results and and Discussion Discussion 3. Results 3.1. Calibration of ALT Biosensors 3.1. Calibration of ALT Biosensors 3.1.1. Sensitivity, Detection Limit, Sampling Time, and Detection Range of ALT Biosensors Tested 3.1.1. Sensitivity, Detection Limit, Sampling Time, and Detection Range of ALT Biosensors Tested by Method 1 by Method 1 For those ALT biosensors tested by Method 1, they were stored at ´20 ˝ C for one day before ForALT those ALT biosensors tested by Method 1, they were (from stored40 atU/L, −20 °C one400 dayU/L, before testing. current responses corresponding to ALT activities 200for U/L, to testing. ALT current responses corresponding to ALT activities (from 40 U/L, 200 U/L, 400 U/L, to 900 U/L) tested by Method 1 were shown in Figure 5a. The detection range is from 40 to 900 U/L 900 U/L) tested by Method 1 wereALT shown in Figure 5a. The detection range is from 40 to 900 U/L and and the range covers the normal concentration presented in the serum and beyond. In Method 1, the range covers the normal ALT concentration presented in the serum and beyond. In Method 1, the the ALT current responses increased as the enzymatic reaction continued and the signal was recorded ALT responses increased thecalibration enzymatic reaction and thetested signalby was recorded for for 60current s (i.e., the sampling time). as The curve ofcontinued ALT biosensors Method 1 was 60 s (i.e., the time). The calibration curve response of ALT biosensors by Method 1 was established by sampling plotting the slope of each ALT current (pA/s) vs. tested ALT activities (U/L) as established by plotting the slope of each ALT current response (pA/s) vs. ALT activities (U/L) as shown in Figure 5b. The sensitivity was defined by the slope of the calibration curve divided by the shown in Figure 5b. The sensitivity was defined by the slope of the calibration curve divided by the electrode area (0.401 mm2 ) of the biosensor. Based on the calibration curve in Figure 5b, the biosensor electrode area mm2)to ofbe the1.908 biosensor. on ˆ the curvemm in Figure the biosensor ´6 nA/(s¨ U/L¨ 2 ). This5b, sensitivity was(0.401 calculated ˆ 10´5Based ˘ 6.739 10calibration sensitivity was sensitivity was calculated to be 1.908 × 10−5 ± 6.739 × 10−6 nA/(s·U/L·mm2). This sensitivity was higher than that presented by Kihara et al. [16], but not as high as that proposed by Cooper et al. [17] and Chang et al. [28]. It is possibly due to the thick permselective layers coated on our ALT biosensors

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higher than that presented by Kihara et al. [16], but not as high as that proposed by Cooper et al. [17] andSensors Chang et al. [28]. It is possibly due to the thick permselective layers coated on our ALT biosensors 2016, 16, 767 7 of 14 which reduce the biosensor sensitivity. To increase the sensor sensitivity, possible solutions may involve thewhich usage reduce of proper derivatives, quinine derivatives, and other organic themediators biosensor including sensitivity.ferrocene To increase the sensor sensitivity, possible solutions may dyes which have been used in the fabrication of biosensors for the detection of glucose [33] or H O involve the usage of proper mediators including ferrocene derivatives, quinine derivatives, and2 other 2 [34] where the redox reaction the mediator coupled with enzymatic to mediate electron organic dyes which haveofbeen used in theisfabrication of biosensors foroxidation the detection of glucose [33] or H2O 2 [34] where redox reaction of the mediator is coupled to mediate transfer between the the active center of enzyme and electrode andwith thusenzymatic enhancesoxidation the electron transfer. electronour transfer the active center better of enzyme and electrode andALT thusagainst enhances the positively electron However, ALTbetween biosensors have much selectivity towards both transfer. However,DA ourand ALT biosensors have much better selectivity towards ALT of against bothof charged interferents negatively charged interferents AA (further discussion the effect positivelywill charged interferents DA and3.2). negatively charged AAand (further discussion ofby the interference be discussed in Section Detection limit interferents was 15.9 U/L was determined the effect of interference will be discussed in Section 3.2). Detection limit was 15.9 U/L and was calibration equation (Equation (6)) obtained in Figure 5), where x is the limit of detection and y is the determined the calibration (Equation (6)) obtained in Figure 5), where is the limit of mean slope of 0byU/L ALT addedequation with two times deviation. Besides, the size of ourxALT biosensors detection and y is the mean slope of 0 U/L ALT added with two times deviation. Besides, the size and of were much smaller than that of other ALT biosensors proposed by others (~125 times, ~4.5 times, our ALT biosensors were much smaller than that of other ALT biosensors proposed by others (~125 times, ~10 times smaller than that proposed by Kihara et al. [16], Cooper et al. [17], and Chang et al. [28], ~4.5 times, and ~10 times smaller than that proposed by Kihara et al. [16], Cooper et al. [17], and respectively) which means the fabrication cost of our ALT biosensors and the reagent volume for Chang et al. [28], respectively) which means the fabrication cost of our ALT biosensors and the reagent testing can be reduced considerably. volume for testing can be reduced considerably. ´3 y “=7.65 x ´−0.156 7.65ˆ 10 10 0.156

(6) (6)

Figure 5. (a) ALT current responses corresponding 200 U/L, U/L,400 400U/L, U/L, Figure 5. (a) ALT current responses correspondingtotoALT ALTactivities activities(from (from40 40U/L, U/L, 200 ˝ C. The to 900 U/L) tested byby Method Thearrows arrowsindicate indicatethe thetiming timingofof to 900 U/L) tested Method1 1after afterone oneday dayofofstorage storageat at´20 −20 °C. addition; The calibration curve ALTbiosensors biosensors(N (N== 10) 10) tested tested by Method ALTALT addition; (b)(b) The calibration curve ofofALT Method1.1.

3.1.2. Sensitivity, Detection Limit, SamplingTime, Time,and andDetection Detection Range Range of of ALT 3.1.2. Sensitivity, Detection Limit, Sampling ALT Biosensors BiosensorsTested Tested by Method 2 by Method 2 determine sensitivitiesofofALT ALTbiosensors biosensors tested tested by by Method Method 2, To To determine sensitivities 2, ALT ALT current currentresponses responses corresponding to ALT activities (from 0 U/L, 10 U/L, 20 U/L, 40 U/L, 70 U/L, 100 corresponding to ALT activities (from 0 U/L, 10 U/L, 20 U/L, 40 U/L, 70 U/L, 100 U/L, U/L,300 300U/L, U/L, 600 U/L, to 900 U/L) were recorded (Figure 6a) and the sensitivity was calculated based on the 600 U/L, to 900 U/L) were recorded (Figure 6a) and the sensitivity was calculated based slope on the of the calibration curve (Figure 6b). It was found that the storage temperature was also an important slope of the calibration curve (Figure 6b). It was found that the storage temperature was also factor which can affect the sensitivity of ALT biosensors. Once fabricated, ALT biosensors were tested an important factor which can affect the sensitivity of ALT biosensors. Once fabricated, ALT after one day of storage at −20 °C and 4 °C. It was shown that ALT biosensors prepared after one day biosensors were tested after one day of storage at ´20 ˝ C and 4 ˝ C. It was shown that ALT biosensors of storage at −20 °C have higher sensitivity and a lower detection limit (0.059 ± 0.029 nA/(U/L·mm2) prepared after one day of storage at ´20 ˝ C have higher sensitivity and a lower detection limit and 8.48 U/L, respectively) compared to that prepared after one day of storage at 4 °C (0.059 ˘ 0.029 nA/(U/L¨ mm22 ) and 8.48 U/L, respectively) compared to that prepared after one day (0.034 ± 0.016 nA/(U/L·mm ) and 14.74 U/L, respectively). The sensitivity was defined by the slope of of storage at 4 ˝ C curve (0.034divided ˘ 0.016by nA/(U/L¨ mm2area ) and(0.401 14.74mm U/L, respectively). was 2) of the calibration the electrode the biosensorThe andsensitivity the detection 2 ) of the biosensor defined by the slope of the calibration curve divided by the electrode area (0.401 mm limit was defined by the ALT concentration corresponding to two times the level of noise. The sensitivity and detection limit of the biosensor prepared by storing at −20 °C was 1.74 times higher than that prepared by storing at 4 °C. This result suggests that a lower storage temperature (−20 °C)

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and the detection limit was defined by the ALT concentration corresponding to two times the level of Sensors 767 of 14 noise. 2016, The 16, sensitivity and detection limit of the biosensor prepared by storing at ´20 ˝ C was 1.74 8times

higher than that prepared by storing at 4 ˝ C. This result suggests that a lower storage temperature during the preparation of ALT biosensors may preserve higher enzyme activity of glutamate oxidase (´20 ˝ C) during the preparation of ALT biosensors may preserve higher enzyme activity of glutamate immobilized on the biosensor and therefore, the biosensors have higher sensitivity towards oxidase immobilized on the biosensor and therefore, the biosensors have higher sensitivity towards glutamate, the product of the ALT enzymatic reaction. Besides, our ALT biosensors tested by Method 2 glutamate, the product of the ALT enzymatic reaction. Besides, our ALT biosensors tested by Method 2 have fast response time (~5 s) defined as the time required reaching 95% of the steady-state sensing have fast response time (~5 s) defined as the time required reaching 95% of the steady-state sensing current. Jamal et al. [3] proposed an ALT biosensor with response time ~36 times longer than ours, current. Jamal et al. [3] proposed an ALT biosensor with response time ~36 times longer than ours, resulting in a long testing interval (8000 s), but the sensitivity and detection limit of their sensors were resulting in a long testing interval (8000 s), but the sensitivity and detection limit of their sensors were 2.6 times higher and 2.6 lower than those of sensors proposed in this study. Song et al. [26] proposed 2.6 times higher and 2.6 lower than those of sensors proposed in this study. Song et al. [26] proposed an ALT biosensor in the micro fluidic system with impressive sensitivity (2.69 nA/(U/L·mm22)) and an ALT biosensor in the micro fluidic system with impressive sensitivity (2.69 nA/(U/L¨ mm )) and detection limit (1.3 U/L); however, the detection range of their sensor only covered from 1.3 to 250 U/L detection limit (1.3 U/L); however, the detection range of their sensor only covered from 1.3 to 250 U/L and no information regarding the effect of interference was provided, while severe liver-damaged and no information regarding the effect of interference was provided, while severe liver-damaged patients can have ALT levels elevated to 240–960 U/L [3]. The detection range of ALT biosensors patients can have ALT levels elevated to 240–960 U/L [3]. The detection range of ALT biosensors tested tested by Method 2 in this work is from 10 to 900 U/L and the range covers the normal ALT by Method 2 in this work is from 10 to 900 U/L and the range covers the normal ALT concentration concentration presented in the serum and beyond. presented in the serum and beyond.

Figure 6. (a) ALT current current responses responses corresponding corresponding to to ALT ALTactivities activities(from (from00U/L, U/L, 10 10 U/L, U/L, 20 U/L, U/L, U/L,70 70U/L, U/L,100 100U/L, U/L, 300 U/L, U/L, to 900 of biosensors tested by Method 2 after one 40 U/L, 300 U/L, 600600 U/L, to 900 U/L)U/L) of biosensors tested by Method 2 after one day ˝ C (— gray line) and ´20 ˝ C (— black line). The arrows indicate the timing of ALT day of storage of storage at 4 at °C4 (━ gray line) and −20 °C (━ black line). The arrows indicate the timing of ALT additions; (b) (b) Calibration Calibration curves 10) tested tested by by Method Method 22 after after one one day day of of storage storage additions; curves of of ALT ALT biosensors biosensors (N (N = = 10) ˝ C ( ) and ´20 ˝ C (). in the refrigerator at 4 in the refrigerator at 4 °C (●) and −20 °C (■).

Effect of of Interference Interference 3.2. Effect studies working working on on ALT biosensors, only several several of them have studies studies on the effect For recent studies interference from negatively negatively charged charged AA AA and and UA at low concentrations concentrations [18,23,27,28] [18,23,27,28] and and few of of interference them reported reported the the effect effect of interference interference from from cations. cations. To To increase increase the the selectivity selectivity of of ALT ALT biosensors, biosensors, them the electrode surface was coated with permselective polymer layers overoxidized Ppy and Nafion®® for hindering charged interferents. The schematic structure and the SEM image of these coatings was shown in Figures 1 and 2c, respectively. In this experiment, positively charged dopamine (DA) and shown negatively charged charged ascorbic ascorbic acid acid (AA) (AA) were used as exemplary interferent species. ALT biosensors negatively 40 µM µM H22O O22, ,250 250µM µM AA, AA, and and 500 500 µM µM AA, AA, and bare Pt electrodes were tested with 20 µM H22O22,, 40 sequentially (Figure 7a), and with 20 µM H H22O22,, 40 40 µM µM H22O O22, ,55µM µMDA, DA,and and 10 10 µM µM DA, DA, sequentially sequentially (Figure 7b). 7b). Almost Almostnone noneofofinterferent interferentcurrent current responses tested ALT biosensors found, but responses tested on on ALT biosensors waswas found, but that that tested on bare Pt electrodes showed excessively highand AADA andresponses. DA responses. The sensitivity of2 tested on bare Pt electrodes showed excessively high AA The sensitivity of H2 O H 2O2 tested onbiosensors ALT biosensors also decreased (i.e.,suggesting 58.5%) suggesting that permselective layers tested on ALT also decreased (i.e., 58.5%) that permselective layers modified modified on ALT biosensors could the alsobiosensor lower thesensitivity; biosensoron sensitivity; on theitother hand, it was on ALT biosensors could also lower the other hand, was observed that observed that permselective layers modified ALT biosensors have notably reduced noise signals. The interferent sensitivities of ALT biosensors towards AA and DA were reduced 3826 and 44.6 times, respectively, compared to those of bare Pt electrodes (see the bar chart shown in Figure 7c). Although these layers may decrease the flux of H2O2 reaching the Pt electrode surface resulting in lower current responses as shown in Figure 7c, the signal noises as well as the interferent currents decreased

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permselective layers modified ALT biosensors have notably reduced noise signals. The interferent sensitivities of ALT biosensors towards AA and DA were reduced 3826 and 44.6 times, respectively, compared to those of bare Pt electrodes (see the bar chart shown in Figure 7c). Although these layers may decrease the flux of H2 O2 reaching the Pt electrode surface resulting in lower current responses Sensors 2016, 16, 767 9 of 14 as shown in Figure 7c, the signal noises as well as the interferent currents decreased significantly as shown in Figure The concentration AA presented in the cerebrospinal fluid is usually significantly as7a,b. shown in Figure 7a,b. Theofconcentration of AA presented in the cerebrospinal fluidhigh is (100–500 µM) [35,36] and the mean concentration of AA presented in the serum usually ranges from usually high (100–500 µM) [35,36] and the mean concentration of AA presented in the serum usually ~30–50 µMfrom [35].~30–50 The concentration of DA rangesoffrom ~0.01–1from µM ~0.01–1 in the extracellular fluid in the ranges µM [35]. The concentration DA ranges µM in the extracellular fluid in thebut brain [36,37], but it wasinrarely found blood in the [38–40]; healthy however, blood [38–40]; however, DA mayinbe brain [36,37], it was rarely found the healthy DA may be presented the presented in level the blood at a high if injectedBesides, as a medication. Besides, the presence some cationic blood at a high if injected as level a medication. the presence of some cationicofpharmaceuticals pharmaceuticals (e.g.,may acetaminophen) may interfere the of sensing of ALT biosensors [18]. (e.g., acetaminophen) also interfere thealso sensing signal ALT signal biosensors [18]. In summary, ® ® In summary,polymer permselective layers,Ppy, overoxidized Ppy,modified and Nafion modified on ALT permselective layers, polymer overoxidized and Nafion on ALT biosensors were biosensors were to be ablecharged to rejectinterferents common charged interferents AA and effectively. In shown to be able toshown reject common AA and DA effectively. In DA Table 1, the figures Table 1, the figures of merit of recently reported electrochemical ALT biosensors are compared. of merit of recently reported electrochemical ALT biosensors are compared.

Figure 7. The effect of interference on Pt bare Pt electrodes and ALT biosensors. (a) Current Figure 7. The effect of interference testedtested on bare electrodes and ALT biosensors. (a) Current responses 2O2 (the of injection H2O2 is indicated by responses upon sequential of 20 µM H upon sequential additions ofadditions 20 µM and 40 µM µM and H2 O40 injection H2 O2 of is indicated by the solid 2 (the thearrow) solid black and 250500 µMµM andAA 500(the µM injection AA (the injection AA is indicated the dashed black and arrow) 250 µM and of AA isofindicated by theby dashed black black arrow) arrow) tested bare Pt electrodes (━and grayALT line)biosensors and ALT biosensors (━ The blackinset line).plot Theshows inset plot tested on bare Pton electrodes (— gray line) (— black line). lower shows lower range; (b) Current responses additions upon sequential additions of 20 and injection 40 µM current range; (b)current Current responses upon sequential of 20 µM and 40 µM H2µM O2 (the H2O O2 (the injection of H2O2 is indicated by the solid black arrow) and 5 µM and 10 µM DA (the injection of H 2 2 is indicated by the solid black arrow) and 5 µM and 10 µM DA (the injection of DA is indicated of is indicated by the dashed blackPtarrow) tested(— on gray bareline) Pt electrodes (━ gray line) ALT by theDA dashed black arrow) tested on bare electrodes and ALT biosensors (—and black line); biosensors (━ black line); (c) Comparison of interferent current responses tested on bare Pt electrodes (c) Comparison of interferent current responses tested on bare Pt electrodes and ALT biosensors (N = 5). and ALT biosensors (N = 5).

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Table 1. Recently reported electrochemical biosensors for the detection of alanine aminotransferase and their figures of merit. No.

Electrode/size (mm2 )

EC Method

Modification & Enzyme Layers

Selectivity

Response Time (s)

Sensitivity

LOD/range

Ref.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

O2 electrode/490.9 Pt/50.3 BIA membrane/28.3 PAC/1.77 Pt/0.05 Au/7.85 ˆ 10´3 Au/19.63 Pt/0.15 Pt/50.5 Pt/0.15 Pd/4.8 Pt/5.5 Graphite/28.3 Pt/0.401 Pt/0.401

CPA CPA CPA CPA CPA DPV ChA CPA CPA CPA CPA CPA CV CPA CPA

Tf, porous AC & PyOx PVC, CA & PyOx PyOx only PC & GlutOx CA, immobilon IA membrane, PC & GlutOx LDH only SAM mediator-coated layer, anti-ALT Ab & PyOx GlutOx only Nanoporous Si & GlutOx GlutOx only Nf & GlutOx Nf, mediated medium & GlutOx P4AP & PyOx Ppy, Nf & GlutOx Ppy, Nf & GlutOx

NA NA NA NA AA, UA NA NA NA NA AA, UA AA, UA NA AA, UA, Glut, Glu AA, DA AA, DA

120 60 240 300 300 NA NA NA 20 600 120 180 200 60 5

NA 1.46 ˆ 10´5 nA/(s¨ U/L¨ mm2 ) NA 3.77 nA/(s¨ U/L¨ mm2 ) NA 0.122 nA/(s¨ U/L¨ mm2 ) 26.3 nA/(ng/mL) NA 2.69 nA/(U/L¨ mm2 ) NA 2.07 ˆ 10´3 nA/(s¨ U/L¨ mm2 ) 0.153 nA/(U/L¨ mm2 ) NA 1.908 ˆ 10´5 nA/(s¨ U/L¨ mm2 ) 0.059 nA/(U/L¨ mm2 )

0.5/0.5–180 U/L 5/5–1600 U/L 3/6–30000 U/L 5/5–500 U/L 2/5–1200 U/L 0.3/0.3–200 U/L 0.01/0.01–1000 ng/mL 11/11–88 U/L 1.3/1.3–250 U/L 9/ 9–250 U/L 8/ 8–250 U/L 3.29/ 10–1000 U/L 2.68ˆ10´5 / 3 ˆ 10´5 –3 U/L 15.9/40–900 U/L 8.48/10–900 U/L

[24] [16] [14] [17] [18] [21] [22] [25] [26] [27] [28] [3] [23] This work a This work b

Abbreviations: Ab (antibody); AC (acetyl-cellulose); BIA (biodyne immunoaffinity); CA (cellulose acetate); ChA (chronoamperometry); CPA (constant potential amperometry); CV (cyclic voltammetry); DPV (differential pulse voltammetry; EC (electrochemical); Glu (glucose); Glut (glutamate); IA (immunoaffinity); NA (not available); Nf (Nafion); P4AP (poly(4-aminophenol)); PAC (platinized activated carbon); PC (polycarbonate); PVC (polyvinyl chloride); SAM (self-assembled monolayer); Tf (Teflon). Superscriptions: a Method 1; b Method 2.

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ALT Biosensors Biosensors 3.3. Stability of ALT enzyme-based amperometric biosensors; for for example, the Many factors factors can canaffect affectthe thestability stabilityofof enzyme-based amperometric biosensors; example, durability of electrodes, thethe method of ofenzyme the durability of electrodes, method enzymeimmobilization, immobilization,the themethod method and and condition condition of storage, etc. [25]. In In this thisexperiment, experiment, the the stability stability of of ALT ALT biosensors biosensors was evaluated based on variations sensorsensitivities sensitivitiesduring duringa aperiod period time. Sensitivities of brand biosensors (Nwere = 5) of sensor of of time. Sensitivities of brand newnew ALTALT biosensors (N = 5) were measured the first day (New), the 14th day2), (Week 2), the day4),(Week 4), and day measured on theon first day (New), the 14th day (Week the 28th day28th (Week and the 56th the day56th (Week 8) ˝ (Week 8) of(in storage (in a dessicant −20the °C)effect and of thestorage effect of storage on sensitivity the sensor of storage a dessicant container container at ´20 C)atand time on thetime sensor sensitivity was investigated. In the Figure 8, thesensitivity relative sensitivity of the ALT biosensor was vs. plotted was investigated. In Figure 8, relative of the ALT biosensor was plotted time vs. of time of measurement show theofability of thetosensor initial sensitivity after aofperiod of measurement to showto the ability the sensor retaintoitsretain initialitssensitivity after a period storage storage time. The sensitivity relative sensitivity of biosensor the ALT biosensor wasasdefined as the sensitivity of the sensor time. The relative of the ALT was defined the sensitivity of the sensor measured measured at certain time divided after storage by the initial sensitivity of the sensor. For ALT at certain time after storage by thedivided initial sensitivity of the sensor. For ALT biosensors tested by biosensors tested by Method 1, of relative sensitivitieswere of ALT biosensors were(Week 100%2), (New), 2), Method 1, relative sensitivities ALT biosensors 100% (New), 122% 171%122% (Week(Week 4), and 171% (Week (Week8), 4),respectively. and 160% (Week 8), respectively. decreasewas in sensor sensitivity was observed after 160% No decrease in sensorNo sensitivity observed after eight weeks of storage. eight weeks of storage. Forby ALT biosensors tested by Methodof2,ALT relative sensitivities ALT(New), biosensors For ALT biosensors tested Method 2, relative sensitivities biosensors were of 100% 104% were 100% (New), (Week 92%8), (Week 4), and 72% respectively. This result showed (Week 2), 92% (Week104% 4), and 72%2), (Week respectively. This(Week result8), showed that ALT biosensors tested thatMethod ALT biosensors tested by Method 2 can retain sensitivities after storage for four weeks by 2 can retain excellent sensitivities after excellent storage for four weeks (relative sensitivity: 92%) (relative sensitivity: 92%) and still good sensitivities after storage for eight weeks and still have good sensitivities afterhave storage for eight weeks (relative sensitivity: 72%). These(relative results ˝ C, and sensitivity: 72%). These resultsetwere comparable Kihara et al. (80% six months of storage at were comparable with Kihara al. (80% after sixwith months of storage at 4after 100% after one year ˝ 4 °C, and 100% after year of storage −20 after °C) [16], al. (83%atafter days of Song storage at of storage at ´20 C)one [16], Cooper et al. at (83% 100Cooper days ofetstorage 4 ˝ C)100 [17], and et al. ˝ C) [26]. 4 °C) after [17], four and months Song et of al. storage (85% after months of storage at 4 °C) [26]. Overall, ALT biosensors (85% at 4four Overall, ALT biosensors tested by both methods can tested bygood bothstorage methods can provide good storage to twosensitivities months. Inof addition, relative provide stability up to two months. Instability addition,up relative ALT biosensors sensitivities of ALT biosensors repeatedly intwo the months 900 U/Lto ALT solution over two months were tested repeatedly in the 900were U/Ltested ALT solution over evaluate their reproducibility. to evaluate reproducibility. When ALT biosensors tested byof Method 1, relative sensitivities When ALT their biosensors were tested by Method 1, relativewere sensitivities ALT biosensors were 100% of ALT139% biosensors were 100% (New), 139% (Week (Week and 173% (Week 8).by When ALT (New), (Week 2), 190% (Week 4), and 173% (Week2),8).190% When ALT 4), biosensors were tested Method 2, biosensors were tested by Method 2, their relative sensitivities were 100% (New), 94% (Week 2), 89% their relative sensitivities were 100% (New), 94% (Week 2), 89% (Week 4), and 80% (Week 8). It showed (Week 4), biosensors and 80% (Week showed that ALT biosensors tested2inprovided the 900 better U/L ALT solution by that ALT tested8).inItthe 900 U/L ALT solution by Method reproducibility Method 2 provided better reproducibility over two months compared to those tested by Method 1. over two months compared to those tested by Method 1.

Figure 8. 8. The storage storage stability stability of ALT ALT biosensors biosensors tested tested by by both both methods. methods. The relative relative sensitivity sensitivity of ALT ALT biosensors biosensors (N (N == 5) was plotted plotted vs. vs. time of measurement.

3.4. Determination of ALT Activities Activities in in Spiked Spiked Samples Samples 200200 U/L, 400 400 U/L,U/L, and 900 Spiked samples samples with withdifferent differentALT ALTactivities activities(20 (20U/L, U/L, U/L, andU/L) 900 were U/L)tested were by bothbyMethod 1 and Method 2. The experimental ALT activities measured by Method 1 were 14 U/L, tested both Method 1 and Method 2. The experimental ALT activities measured by Method 1 were 180U/L, U/L,180 408U/L, U/L,408 andU/L, 959 and U/L 959 andU/L the and recoveries were found be 70%, 107% 14 the recoveries were to found to be90%, 70%, 102%, 90%, 102%, corresponding to spiked ALT activities (20 U/L, 200 U/L, 400 U/L, and 900 U/L), respectively. The

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107% corresponding to spiked ALT activities (20 U/L, 200 U/L, 400 U/L, and 900 U/L), respectively. experimental ALT activities measured by by Method 2 were 21 U/L, 201 201 U/L,U/L, 355 U/L, and 663 The experimental ALT activities measured Method 2 were 21 U/L, 355 U/L, andU/L 663and U/L the recoveries were found to be 103%, 101%, 89%, 74% corresponding to spiked ALT activities and the recoveries were found to be 103%, 101%, 89%, 74% corresponding to spiked ALT activities U/L,200 200U/L, U/L,400 400U/L, U/L, and and 900 900 U/L), U/L), respectively. ALT (20(20 U/L, respectively.The Theresults resultsofofmeasured measuredexperimental experimental ALT activitieswere wereshown shownininFigure Figure9.9. Based Based on on the the results, results, it activities it suggested suggestedthat thatMethod Method11provides providesbetter better estimationfor fordetermining determiningALT ALTactivities activitiesatathigher higher activity activity range range (200~900 (200~900 U/L), estimation U/L),while whileMethod Method2 2 provides better estimation for determining ALT activities at lower activity range (20~400 U/L). provides better estimation for determining ALT activities at lower activity range (20~400 U/L).

Figure 9. 9.Experimental ♦)and andMethod Method22(■) ()atatspiked spikedALT ALT Figure ExperimentalALT ALTactivities activities measured measured by by Method Method 1 1 ((◊) activities: 2020 U/L, U/L,and and900 900U/L U/L = 5). inset shows lower activity range. activities: U/L,200 200U/L, U/L, 400 U/L, (N(N = 5). TheThe inset plotplot shows lower activity range.

Conclusions 4. 4.Conclusions convenient, selective biosensor on platinum wire microelectrode AA convenient, fast,fast, andand selective ALT ALT biosensor based based on platinum wire microelectrode modified ® and immobilized by GlutOx has been demonstrated successfully. ® modified by Ppy and Nafion by Ppy and Nafion and immobilized by GlutOx has been demonstrated successfully. Compared to Compared other studies working on ALT biosensors, ourprovide sensors the can best provide the bestagainst selectivity other studiesto working on ALT biosensors, our sensors can selectivity both against both negatively and positively charged interferents, the fastest response time (~5 s), good negatively and positively charged interferents, the fastest response time (~5 s), good storage stability storage eight weeks, good U/L), reasonable (70%–107%) over eightstability weeks,over good detection rangedetection (10–900range U/L),(10–900 reasonable recoveriesrecoveries (70%–107%) in spiked 2)) and detection limit (8.48 U/L); in in spiked ALT samples, and fair sensitivity (0.059 nA/(U/L·mm 2 ALT samples, and fair sensitivity (0.059 nA/(U/L¨ mm )) and detection limit (8.48 U/L); in addition, addition, the ultra-small size of the sensor allows economic production of the ALT biosensor as well the ultra-small size of the sensor allows economic production of the ALT biosensor as well as reducing as reducing the volume of testing reagents required. The proposed ALT biosensor may be used for the volume of testing reagents required. The proposed ALT biosensor may be used for assisting the assisting the development of a convenient diagnosing technique for liver diseases. development of a convenient diagnosing technique for liver diseases. Acknowledgments: Financial support by the Ministry of Science and Technology (Taiwan) under grant numbers Acknowledgments: Financial support by the Ministry of Science and Technology (Taiwan) under grant numbers MOST 104-2221-E-011-145and and by by National National Taiwan Taiwan University University of MOST 104-2221-E-011-145 of Science Science and and Technology—NTU Technology—NTUTriangle Triangle Alliance—Young Researcher InnovativeCollaborative CollaborativeGrant Grant(105TY6A068) (105TY6A068) are are gratefully Alliance—Young Researcher Innovative gratefully acknowledged. acknowledged. Author Contributions:Tran Tran Nguyen performed her her MScMSc thesis working in this in research. Tina T.-C. Tseng Author Contributions: NguyenThanh ThanhThuy Thuy performed thesis working this research. Tina T.-C. Tseng supervised the research and contributed the writing, design, and analysis the All data. All authors read supervised the research and contributed in theinwriting, design, and analysis of the of data. authors read and and approved final manuscript. approved thethe final manuscript.

Conflicts of of Interest: The authors Conflicts Interest: The authorsdeclare declareno noconflict conflictof ofinterest. interest.

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