membranes

Review Application of Semipermeable Membranes in Biosensing

Tanmay Kulkarni and Gymama Slaughter * Bioelectronics Laboratory, Department of Computer Science and Electrical Engineering, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-410-455-8483

Academic Editor: Hsueh-Chia Chang Received: 30 October 2016; Accepted: 8 December 2016; Published: 14 December 2016

Abstract: Glucose have received significant attention in recent years due to the escalating mortality rate of diabetes mellitus. Although there is currently no cure for diabetes mellitus, individuals living with diabetes can lead a normal life by maintaining tight control of their blood glucose levels using glucose biosensors (e.g., glucometers). Current research in the field is focused on the optimization and improvement in the performance of glucose biosensors by employing a variety of glucose selective , mediators and semipermeable membranes to improve the electron transfer between the active center of the and the substrate. Herein, we summarize the different semipermeable membranes used in the fabrication of the glucose , that result in improved biosensor sensitivity, selectivity, dynamic range, response time and stability.

Keywords: glucose biosensor; semipermeable membrane; glucose oxidase; pyrolloquinoline quinone glucose dehydrogenase; polypyrrole; poly(3,4-ethylenedioxythiophene; cellulose acetate

1. Introduction Diabetes mellitus is the seventh leading cause of death in the US. Diabetes is broadly classified into two types: type I and type II. Type I diabetes is a result of insufficient insulin production by the pancreas, whereas type II diabetes is due to the body’s inability to use the insulin that is produced, hence the name insulin resistance is used to refer to type II diabetes. Currently, individuals with diabetes are able to monitor their blood glucose levels using a glucometer or a continuous glucose monitor (CGM) in order to prevent further complications such as blindness, ketoacidosis, stroke and even amputation. While the National Institute of Health, the American Diabetes Association and Centers for Disease Control and Prevention are working closely together to find a cure for diabetes, several approaches to “cure” diabetes have been proposed. Some of these approaches include pancreas transplantation, islet cell transplantation, artificial pancreas development and genetic manipulation [1–4]. These approaches are still in their early stages and possess a lot of challenges. Blood glucose monitoring on a timely basis is the current optimal solution to keep blood glucose levels under control. Blood glucose monitors consist of a glucose transducer and electronics that display blood glucose level information in mg/dL. The glucose transducer is an analytical device that converts the chemical energy in glucose to electrical energy and, when coupled with a potentiostat circuit, it is then capable of measuring and displaying the glucose concentration in blood. These traditional glucose monitors consist of a potentiostat circuit which is battery operated, thereby making blood glucose monitors bulky. Various glucose biosensors are available on the market today, which mostly operate based on the principles of coulometric or amperometric electrochemical detection methods [5]. While the coulometric principle relies on the measurement of the total charge necessary to oxidize a finite amount of glucose, the amperometric principle measures the steady state current produced from a finite volume of glucose being oxidized. Typically, a columetric-based biosensor employs a test strip as depicted in

Membranes 2016, 6, 55; doi:10.3390/membranes6040055 www.mdpi.com/journal/membranes Membranes 2016, 6, 55 2 of 20 Membranes 2016, 6, 55 2 of 20 from a finite volume of glucose being oxidized. Typically, a columetric-based biosensor employs a test strip as depicted in Figure 1, consisting of a fill test electrode that fills the test strip with the Figureglucose1 ,substrate consisting, which of a fill is testthen electrode oxidized that by fillsa glucose the test-selective strip with enzyme the glucose and the substrate, amount of which charge is thenrequired oxidized to oxidize by a glucose-selective the glucose substrate enzyme isand measured the amount when of charge a potential required is toapplied oxidize between the glucose the substrateworking and is measured the reference when electrode a potential via is appliedthe battery between operated the working potentiostat. and the The reference measured electrode charge via is theproportional battery operated to the glucose potentiostat. concentration. The measured charge is proportional to the glucose concentration.

FigureFigure 1. 1.Glucose Glucose biosensingbiosensing principles.principles. (A) Coulometric;Coulometric; and and ( (BB)) Am Amperometricperometric glucose glucose biosensor biosensor..

On the other hand, the amperometric-based glucose biosensor uses a glucose selective enzyme On the other hand, the amperometric-based glucose biosensor uses a glucose selective enzyme at at the working electrode to oxidize the glucose, which results in the release of electrons. The steady the working electrode to oxidize the glucose, which results in the release of electrons. The steady state state current is measured by applying a potential between the working electrode and the reference current is measured by applying a potential between the working electrode and the reference electrode electrode to decompose the hydrogen peroxide produced by the oxidation of glucose. Blood volume to decompose the hydrogen peroxide produced by the oxidation of glucose. Blood volume as small as small as 0.3 µL is sufficient for glucose sensing. Since blood glucose monitoring via the use of as 0.3 µL is sufficient for glucose sensing. Since blood glucose monitoring via the use of glucometers glucometers requires frequent finger pricking, this can be tedious and painful at times. A completely requires frequent finger pricking, this can be tedious and painful at times. A completely non-invasive non-invasive GlucoWatch® G2 Biographer (Cygnus, Redwood City, CA, USA) glucose monitoring GlucoWatch® G2 Biographer (Cygnus, Redwood City, CA, USA) glucose monitoring device relies device relies on reverse iontophoresis principle for measuring glucose levels. It measures glucose in on reverse iontophoresis principle for measuring glucose levels. It measures glucose in interstitial interstitial fluid. The negative charge of the skin at buffered pH causes it to be permselective to cations fluid. The negative charge of the skin at buffered pH causes it to be permselective to cations such such as sodium and potassium , allowing iontophoresis that causes electroosmosis, by which as sodium and potassium ions, allowing iontophoresis that causes electroosmosis, by which neutral neutral molecules, including glucose, are transported across the skin. However, due to the molecules, including glucose, are transported across the skin. However, due to the discrepancies in the discrepancies in the glucose readings resulting from the interference of sweat, the use of GlucoWatch glucose readings resulting from the interference of sweat, the use of GlucoWatch was discontinued, was discontinued, leaving the glucometer and CGM devices as the most commonly used glucose leaving the glucometer and CGM devices as the most commonly used glucose biosensors for blood biosensors for blood glucose monitoring. glucose monitoring. Although there has been a significant progress in the development of glucose that are Although there has been a significant progress in the development of glucose sensors that are more more compact and easy to use, drawbacks such as calibration issues, bulkiness of the device, warm- compact and easy to use, drawbacks such as calibration issues, bulkiness of the device, warm-up period up period and the dependence on battery to drive the potentiostat circuit still remain. Therefore, and the dependence on battery to drive the potentiostat circuit still remain. Therefore, significant significant research is underway to design novel glucose biosensors that are self-powered [6]. Focus research is underway to design novel glucose biosensors that are self-powered [6]. Focus is on is on improving the sensing parameters using various glucose selective enzymes along with the use improving the sensing parameters using various glucose selective enzymes along with the use of of mediators and semipermeable membranes. In addition, the use of semipermeable membrane has mediators and semipermeable membranes. In addition, the use of semipermeable membrane has gained considerable attention due to its advantages in improving the sensitivity and selectivity of gained considerable attention due to its advantages in improving the sensitivity and selectivity of glucose biosensors. Unlike mediators, which improve the electron transfer between the enzyme and glucose biosensors. Unlike mediators, which improve the electron transfer between the enzyme and the the substrate at the cost of complexity and selectivity. Glucose biosensors with semipermeable substrate at the cost of complexity and selectivity. Glucose biosensors with semipermeable membranes membranes have been demonstrated to enhance the dynamic range along with sensitivity and have been demonstrated to enhance the dynamic range along with sensitivity and selectivity [7]. selectivity [7]. The semipermeable membrane encapsulates the enzyme as well as provides a The semipermeable membrane encapsulates the enzyme as well as provides a microenvironment microenvironment conducive to maintaining the enzyme’s viability, thereby improving the stability conducive to maintaining the enzyme’s viability, thereby improving the stability of glucose biosensors. of glucose biosensors. The first glucose biosensor with a semipermeable membrane was developed The first glucose biosensor with a semipermeable membrane was developed in 1962 by Leland Clark in 1962 by Leland Clark and Champ Lyons [8]. Figure 2 depicts the schematic illustration of the and Champ Lyons [8]. Figure2 depicts the schematic illustration of the glucose biosensor composed of glucose biosensor composed of an oxygen electrode, an inner oxygen semipermeable membrane, a an oxygen electrode, an inner oxygen semipermeable membrane, a thin layer of glucose oxidase (GOx) thin layer of glucose oxidase (GOx) as the glucose selective enzyme, and an outer dialysis membrane. as the glucose selective enzyme, and an outer dialysis membrane. It measures the decrease in oxygen It measures the decrease in oxygen concentration, which is directly proportional to the glucose concentration, which is directly proportional to the glucose concentration. Further simplifications in the concentration. Further simplifications in the design of this particular glucose biosensor were made design of this particular glucose biosensor were made by Updike and Hicks, wherein they immobilized by Updike and Hicks, wherein they immobilized GOx in a polyacrylamide gel on the oxygen GOx in a polyacrylamide gel on the oxygen electrode in order to create a microenvironment that electrode in order to create a microenvironment that stabilizes GOx [9,10]. stabilizes GOx [9,10].

Membranes 2016, 6, 55 3 of 20 Membranes 2016, 6, 55 3 of 20

Figure 2. The first first oxygen electrodeelectrode developed by Clark in 1962.1962.

2. Generations of Glucose Biosensors 2. Generations of Glucose Biosensors Figure 3 provides a schematic diagram of the various generations of glucose biosensors. First- Figure3 provides a schematic diagram of the various generations of glucose biosensors. generation glucose biosensors consist of a natural oxygen substrate and rely upon the detection of First-generation glucose biosensors consist of a natural oxygen substrate and rely upon the detection hydrogen peroxide production, which is directly proportional to the glucose concentration. The of hydrogen peroxide production, which is directly proportional to the glucose concentration. major advantage of the first-generation glucose biosensor is that the measurement process is The major advantage of the first-generation glucose biosensor is that the measurement process is straightforward [11]. However, a huge drawback of these devices is the performance of the biosensor straightforward [11]. However, a huge drawback of these devices is the performance of the biosensor in the presence of interfering species. Hydrogen peroxide is decomposed at a potential of 700 mV, at in the presence of interfering species. Hydrogen peroxide is decomposed at a potential of 700 mV, which other interfering species such as ascorbic acid and uric acid present in blood are also readily at which other interfering species such as ascorbic acid and uric acid present in blood are also readily decomposed [12]. This greatly reduces the selectivity of the glucose biosensors and often results in decomposed [12]. This greatly reduces the selectivity of the glucose biosensors and often results in false false glucose readings. Moreover, the second-generation glucose biosensors rely heavily on glucose readings. Moreover, the second-generation glucose biosensors rely heavily on mediators and mediators and overcome some of the drawbacks of the first generation glucose biosensors. Mediators overcome some of the drawbacks of the first generation glucose biosensors. Mediators are chemical are chemical species that carry electrons from the active centers of the enzyme to the electrode. Some species that carry electrons from the active centers of the enzyme to the electrode. Some of the common of the common mediators employed are ferrocene, thionine and methylene blue. While the mediators mediators employed are ferrocene, thionine and methylene blue. While the mediators are reduced are reduced when the glucose oxidation reaction proceeds, these reduced mediators are then re- when the glucose oxidation reaction proceeds, these reduced mediators are then re-oxidized at the oxidized at the surface of the electrode thus, providing an amperometric output signal and the re- surface of the electrode thus, providing an amperometric output signal and the re-oxidized mediators oxidized mediators can be reused in the reaction [13–16]. Further, by “electrically wiring” can be reused in the reaction [13–16]. Further, by “electrically wiring” redox polymer to the enzymes, polymer to the enzymes, electrons could be shuttled from the enzymes to the transducer [17]. In spite electrons could be shuttled from the enzymes to the transducer [17]. In spite of the use of mediators, of the use of mediators, these glucose biosensors still suffer from the effects of interfering species such these glucose biosensors still suffer from the effects of interfering species such as ascorbic acid and as ascorbic acid and uric acid. Along with the toxicity of some of the mediators, biocatalytic uric acid. Along with the toxicity of some of the mediators, biocatalytic destruction of potential destruction of potential interferences has been observed [18–20]. In order to overcome the interferences has been observed [18–20]. In order to overcome the complications and complexity of complications and complexity of employing mediators in biosensor design and development, third- employing mediators in biosensor design and development, third-generation glucose biosensors were generation glucose biosensors were developed. These biosensors are mediatorless and employ direct developed. These biosensors are mediatorless and employ direct electron transfer mechanism between electron transfer mechanism between the active center of the enzymes and the electrode surface. In the active center of the enzymes and the electrode surface. In these biosensors, a highly conductive these biosensors, a highly conductive substrate is modified such that there is no need for a mediator. substrate is modified such that there is no need for a mediator. However, only a few enzymes such as However, only a few enzymes such as pyroloquinoline quinone glucose dehydrogenase (PQQ-GDH) pyroloquinoline quinone glucose dehydrogenase (PQQ-GDH) and GOx have been reported to achieve and GOx have been reported to achieve direct electron transfer [21–23]. These biosensors have been direct electron transfer [21–23]. These biosensors have been miniaturized using novel microfabrication miniaturized using novel microfabrication technology and thus, found their application in in vivo technology and thus, found their application in in vivo blood glucose monitoring. blood glucose monitoring.

Membranes 2016, 6, 55 4 of 20 Membranes 2016, 6, 55 4 of 20

Figure 3. A schematic of the reactions governing different glucose generations [[23]23]..

In order to develop an optimal and efficient glucose biosensor, it is important to understand the In order to develop an optimal and efficient glucose biosensor, it is important to understand the following eight key characteristics of a glucose biosensor: following eight key characteristics of a glucose biosensor: 1. Accuracy: The ability of the glucose biosensor to produce the output reading close to the true 1. Accuracy: The ability of the glucose biosensor to produce the output reading close to the true value. The sensor needs to be accurate since any discrepancies can result in drifted glucose value. The sensor needs to be accurate since any discrepancies can result in drifted glucose readings, which can prove to be fatal. readings, which can prove to be fatal. 2. Sensitivity: Sensitivity is determined from the slope of the calibration curve. It is a measure of 2. Sensitivity: Sensitivity is determined from the slope of the calibration curve. It is a measure of the change in the biosensor’s output current over the change in glucose concentration. An ideal the change in the biosensor’s output current over the change in glucose concentration. An ideal biosensor will exhibit high and constant sensitivity. biosensor will exhibit high and constant sensitivity. 3. Selectivity: Blood is a complex matrix and consists of components other than glucose. 3. InterferenceSelectivity: Blood species is asuch complex as ascorbic matrix acid and consistsand uric of acid components and competing other than species glucose. such Interferenceas fructose, xylose,species sucrose, such as ascorbic and galactose acid and are uric present acid andin the competing body. An species ideal glucose such as biosensor fructose, xylose, will be sucrose, highly selectiveand galactose towards are presentglucose incompared the body. to An the ideal other glucose interfering biosensor and willcompeting be highly species. selective towards 4. Dynamicglucose compared range: An to ideal the otherglucose interfering biosensor and must competing have a wide species. dynamic range. Dynamic range is 4. definedDynamic as range: the range An idealof glucose glucose concentration biosensor must over have which a wide the sensor dynamic produces range. linear Dynamic response. range isIt isdefined essentia asl thefor rangea glucose of glucose sensor concentrationto detect hypoglycemic over which glucose the sensor (<70 producesmg/dL) levels linear as response. well as hyperglycemicIt is essential for glucose a glucose (>100 sensor mg/dL) to detect levels hypoglycemic along with glucose the normal (<70 mg/dL) glucose levels levels as (70 well–100 as mg/dL).hyperglycemic glucose (>100 mg/dL) levels along with the normal glucose levels (70–100 mg/dL). 5. TestingTesting volume:volume: FromFromthe the point point of of view view of of patient patient convenience, convenience an, idealan ideal biosensor biosensor should should be able be ableto operate to operate with a with minimal a minimal amount amount of blood of sample. blood sample. Initial glucose Initial biosensorglucose biosensor designs required designs requiredapproximately approximately 30 µL of blood. 30 µL But of with blood. advancement But with in microtechnology advancement in and microtechnology improvement in and the improvementdesign of glucose in the biosensors, design of total glucose volume biosensors, of blood required total volume for testing of bloo is presentlyd required as lowfor testing as 0.3 µ isL. 6. presentlyResponse as time: low Anas 0.3 ideal µL. biosensor should have fast response time. The response time varies 6. Responsefor various time: glucose An ideal biosensors. biosensor It ranges should between have fast 3 response and 60 s. time. Since The the glucoseresponse concentration time varies for is variousproportional glucose to the biosensor steadys state. It ranges current, between it is essential 3 and for 60 as sensor. Since to the reach glucose steady concentration state response is proportionalas quickly as to possible. the steady state current, it is essential for a sensor to reach steady state response 7. asCalibration: quickly as possible. This is a very important characteristic of a glucose biosensor. It is a measure of 7. Calibration:the stability This of the is glucosea very important biosensor. characteristic An ideal glucose of a glucose biosensor biosensor. should It not is a require measure frequent of the stabilityrecalibration. of the It glucose should be biosensor. able to detect An idea glucosel glucose for days, biosensor sometimes should up not to require months frequent without recalibration.recalibration. ItHowever, should becurrent able glucose to detect biosensors glucose for need days, calibration sometimes whenever up to new months batch without of test recalibration.strips are used. However, current glucose biosensors need calibration whenever new batch of test 8. stripsSpecificity: are used. This refers to the ability of the glucose biosensor to correctly determine the glucose 8. Specconcentrationificity: This in refers the blood to the sample. ability The of the choice glucose of enzyme biosensor plays to an correctly important determine role in determining the glucose concentrationthe specificity in of the the blood glucose sample biosensor.. The Atchoice times, of enzyme the enzyme plays will an be important specific torole certain in determining functional thegroup specificity instead of of the an glucose individual biosensor. analyte. At An times, ideal the glucose enzyme sensor will willbe specific have high to certain specificity. functional group instead of an individual analyte. An ideal glucose sensor will have high specificity. Due to the inconveniences of the present glucose monitoring devices, self-powered glucose biosensorsDue to are the being inconveniences developed [ 6 of,24 the]. The present success glucose of such monitoring systems depend devices, upon self glucose-powered biosensors glucose thatbiosensors are of are the being best quality.developed One [6,24 of]. theThe factorssuccess enhancing of such systems glucose depend biosensor upon keyglucose characteristics biosensors arethat semipermeableare of the best quality. membranes One employedof the factors to encapsulateenhancing glucose enzymes biosensor as well key as characteristics to screen against are interferingsemipermeable species. membranes employed to encapsulate enzymes as well as to screen against interfering species.

MembranesMembranes 2016 2016,, 6, ,6, 55, 55 55 55 5 of of 20 20 20

3.3. Semi Semippermeableermeable Membranes Membranes 3. Semipermeable Membranes TheThe semipermeable semipermeable membrane membrane used used in in biosensing biosensing applications applications is is a a biological biological or or synthetic synthetic membraneThe semipermeable that allows preferential membrane passage used of in certain biosensing analytes applications (e.g., molecules is a biological and ions) based or synthetic on size membraneMembranes that 2016allows, 6, 55 preferential passage of certain analytes (e.g., molecules and ions) based5 of 20 on size andmembraneand/or/or net net thatchargecharge allows via via diff preferentialdiffusion.usion. This This passage t herebythereby of certain limit limitss analytesthe the diffusion diffusion (e.g., moleculesof of unwanted unwanted and analyte analyte ions) basedss that that on could could size potentiallyand/orpotentially3. net Semi interfere charge interferepermeable via with with diffusion. Membranes thethe desired desired This thereby chemical chemical limits reactions reactions the diffusion with with thethe of unwanted biorecognition biorecognition analytes element element that could (e.g., (e.g., enzyme)potentiallyenzyme) as as interfereshownThe shown semipermeable in in with Figure Figure the 4. desired 4. membraneMany Many chemicalfactors factors used such insuch reactions biosensing as as the the withconcentration concentration applications the biorecognition isof of a the the biological analyte analyte element or; ; s syntheticsize,ize, (e.g., net net enzyme) charge, charge, membraneasmembrane shownmembrane in pore Figurepore size, thatsize,4. Manyallowstemperature temperature factorspreferential suchand and passage pressure aspressure the of concentration certain govern govern analytes the the ofpassage (e.g.,passage the molecules analyte; of of the the size,and analyte analyte ions) net charge,based[25] [25]. .onPermeability Permeability membranesize ofporeof the the size,membrane membraneand temperature/or net chargeplays plays avia anda significant significant diff pressureusion. Thisrole role govern tinhereby in prohibiting prohibiting the limit passages the pass passdiffusion ofageage the of of analyte undesired unwantedundesired [25 analyte molecular]. molecular Permeabilitys that and/could and/or or of ionic ionic the species.membranespecies. potentiallyThis This plays is is governed governed a interfere significant by withby the role thethe membrane inmembrane desired prohibiting chemical chemical chemical passage reactions property, property, of undesired with thethickness thickness biorecognition molecular and and pore and/or pore element size. size.ionic (e.g., A A glucose species.glucose enzyme) as shown in Figure 4. Many factors such as the concentration of the analyte; size, net charge, biosensorThisbiosensor is governed operating operating by thein in vivo vivo membrane is is often oftenchemical subject subject to to property, a a variety variety thickness of of chemical chemical and species porespecies size. in in plasma Aplasma glucose that that biosensor can can foul foul membrane pore size, temperature and pressure govern the passage of the analyte [25]. Permeability operating in vivo is often subject to a variety of chemical species in plasma that can foul and/or interfere andand/or/or interfere ofinterfere the membrane with with the theplays chemical chemical a significant reaction reaction role in occurring prohibitingoccurring on passon the theage active ofactive undesired site site of ofmolecular the the bioelectrode bioelectrode and/or ionic, ,there therebyby resultingwithresulting thespecies. chemical in in a a Thisdecrease decrease reaction is governed in inoccurring the the by selectivity selectivitythe onmembrane the active of of chemicalthe the site biosensor biosensor of property, the bioelectrode, [26] [26] thickness. . Additionally, Additionally, therebyand pore resulting size. biosensors biosensors A glucose in a decrease employ employ semipermeableinsemipermeable the selectivitybiosensor membrane operatingmembrane of the biosensor ins s vivoto to prohibit prohibitis [ 26often]. Additionally, subjectthese these competing tocompeting a variety biosensors ofand and chemical non non employ-competing- competingspecies semipermeable in plasma analytes analytes that from can membranesfrom foul diffusing diffusing to throughprohibitthroughand these to to/ or the the interfere competing biorecognition biorecognition with andthe chemical non-competing element, element, reaction thereby, thereby, occurring analytes allowing allowing on from the active diffusing the the passagesitepassage of through the and bioelectrode and detection to detection the, biorecognition there of ofby glucose glucose resulting in a decrease in the selectivity of the biosensor [26]. Additionally, biosensors employ moleculeselement,molecules thereby, only. only. The The allowing decomposition decomposition the passage of of andascorbic ascorbic detection acid acid at ofat 700 glucose700 mV mV results moleculesresults in in the only.the release release The decomposition of of two two electrons electrons of semipermeable membranes to prohibit these competing and non-competing analytes from diffusing ascorbicand corresponding acid at 700 mVpositive results ions, in the which release affect of twothe electronsmeasured and current corresponding, which often positive leads ions,to incorrect which and correspondingthrough to the positive biorecognition ions, which element, affect thereby, the measured allowing the current passage, which and detection often leads of glucose to incorrect affect the measured current, which often leads to incorrect glucose readings. Thus, semipermeable glucoseglucose moleculesreadings. readings. only. Thus, Thus, The semipermeable semipermeabledecomposition of membranes ascorbicmembranes acid areat are 700 necessary necessary mV results to toin improve improvethe release the theof twoselectivity selectivity electrons of of these these glucosemembranesglucose andbiosensors. biosensors. corresponding are necessary Permselective Permselective positive to improve ions, membranes membranes which the affect selectivity such suchthe measured as as cellulose ofcellulose these current acetate, glucoseacetate,, which polyaniline, polyaniline, biosensors.often leads to polypyrrole polypyrroleincorrect Permselective are are basedmembranesbased on onglucose size size such exclusion, exclusion,readings. as cellulose Thus, whereas whereas semipermeable acetate, semipermeable semipermeable polyaniline, membranes membranes membranes polypyrrole are necessary such such are to asimprove as based nafi nafion,on, onthe poly(vin size poly(vinselectivity exclusion,ylpyridine)ylpyridine) of these whereas and and poly(estersemipermeablepoly(esterglucose-sulfonic-sulfonic biosensors. membranes acid) acid) arePermselective are suchbased based as on nafion,on membranes charge charge poly(vinylpyridine) exclusion exclusion such as cellulose [27] [27]. .These These acetate, and polymer poly(-sulfonicpolymer polyaniline, films films polypyrrole are are usually usually acid) are are solvent solvent based-- based on size exclusion, whereas semipermeable membranes such as nafion, poly(vinylpyridine) and castoncast charge or or electropolymerized. electropolymerized. exclusion [27]. These Table Table polymer 1 1 provides provides films chemical arechemical usually structures structures solvent-cast of of the the or electropolymerized.mostmost commonly commonly employed employed Table1 poly(ester-) are based on charge exclusion [27]. These polymer films are usually solvent- provides chemical structures of the most commonly employed semipermeable membranes used in semipermeablesemipermeablecast or electropolymerized. membranes membranes used used Table in in biosensing biosensing1 provides chemicalapplications. applications. structures of the most commonly employed biosensingsemipermeable applications. membranes used in biosensing applications.

Figure 4. Basic working principle of semipermeable membrane. Figure 4. Basic working principle of semipermeable membrane. FigureFigure 4. 4. Basic Basic working working principle principle of of semipermeable semipermeable membrane membrane. . Table 1. The chemical structures of commonly used semipermeable membranes in biosensing. Table 1. The chemical structures of commonly used semipermeable membranes in biosensing. TableTable 1. 1. The The chemical chemical structures structures of of commonly commonlyCellulose used used Acetate semipermeable semipermeable membranes membranes in in biosensing. biosensing.

CelluloseCellulose Acetate Acetate

Nafion

NafionNafionNafion

Polypyrrole

PolypyrrolePolypyrrole

Membranes 2016, 6, 55 6 of 20

Table 1. Cont. MembranesMembranes 2016 2016, 6,, 655, 55 6 of6 of 20 20 Membranes 2016, 6, 55 6 of 20 Membranes 2016, 6, 55 Polypyrrole 6 of 20

Polyurethane PolyurethanePolyurethane Polyurethane

ChitosanChitosan Chitosan

Poly(2Poly(2-hydroxyethyl-hydroxyethyl methacrylate) methacrylate) Poly(2-hydroxyethyl methacrylate) Poly(2-hydroxyethylPoly(2-hydroxyethyl methacrylate)

4. Non-Enzymatic Glucose Biosensor Membranes 4.4. Non Non-Enzymatic-Enzymatic Glucose Glucose Biosensor Biosensor Membranes Membranes 4. Non Non-Enzymatic-Enzymatic Glucose Biosensor Membranes NonNon-enzymatic-enzymatic glucose glucose biosensors biosensors employ employ precious precious metals metals as as catalyst catalysts sto to oxidize oxidize glucose. glucose. Non-enzymatic glucose biosensors employ precious metals as catalysts to oxidize glucose. RealizingRealizingNon-enzymatic the the ability ability of glucoseof precious precious biosensors metals metals such employsuch as as platinum, platinum, precious gold, metalsgold, copper copper as catalysts and and palladium palladium to oxidize to to directly glucose. directly Realizing the ability of precious metals such as platinum, gold, copper and palladium to directly electrooxidizeRealizingelectrooxidize the ability glucose, glucose, of significant precious significant metals research research such has has as been platinum, been conducted conducted gold, incopper in fabricating fabricating and palladium glucose glucose biosensors to biosensors directly electrooxidize glucose, significant research has been conducted in fabricating glucose biosensors usingelectrooxidizeusing these these precious precious glucose, metal significant metal catalysts catalysts research [2 [28–8 has31–31]. ] been. Bare Bare conducted metal metal electrodes in fabricating were were glucose initially initially biosensors constructed constructed using as as using these precious metal catalysts [28–31]. Bare metal electrodes were initially constructed as glucosetheseglucose precious sensors. sensors. metal Upon Upon catalysts glucose glucose oxidation [ 28oxidation–31]. Bare in in the metalthe presence presence electrodes of of these these were metal metal initially electrodes, electrodes, constructed it itwas was asobserved observed glucose glucose sensors. Upon glucose oxidation in the presence of these metal electrodes, it was observed thatsensors.that poisonous poisonous Upon metal glucosemetal oxides oxidation were were formed formed in the on presence on the the surface surface of these of of the the metal electrode, electrode, electrodes, which which itaffected affected was observed the the stabil stabil thatityity that poisonous metal oxides were formed on the surface of the electrode, which affected the stability andpoisonousand sensitivity sensitivity metal of ofoxides the the glucose glucose were formed electrode electrode on the[32] [32] surface. Moreover,. Moreover, of the due electrode, due to to limited limited which reserves reserves affected of ofthe these these stability precious precious and and sensitivity of the glucose electrode [32]. Moreover, due to limited reserves of these precious metals,sensitivitymetals, these these of metals themetals glucose became became electrode cost cost-ineffective-ineffective [32]. Moreover, for for use use as due as bioelectrodes. bioelectrodes. to limited reserves As As a aresult, result, of these bi bi-metallic- preciousmetallic catalysts catalysts metals, metals, these metals became cost-ineffective for use as bioelectrodes. As a result, bi-metallic catalysts werethesewere used metals used in in becameplace place of of cost-ineffective mono mono-metallic-metallic for catalysts catalysts use as bioelectrodes.to t ooxidize oxidize glucose glucose As a result,[33,34 [33,34] bi-metallic. ]Although. Althoughcatalysts this this overcame overcame were used the the were used in place of mono-metallic catalysts to oxidize glucose [33,34]. Although this overcame the drawbacksindrawbacks place of mono-metallicof of cost cost-effectiveness-effectiveness catalysts and and to poisonous oxidizepoisonous glucose metal metal [oxide33 ,34]. byproduct Althoughbyproducts this,s sensor, sensor overcame parameters parameters the drawbacks such such as as drawbacks of cost-effectiveness and poisonous metal oxide byproducts, sensor parameters such as sensitivity,ofsensitivity, cost-effectiveness selectivity selectivity and and and poisonous dynamic dynamic metal range range oxide remain remain byproducts, a a challenge. challenge. sensor In parametersIn order order to to suchimprove improve as sensitivity, the the key key sensitivity, selectivity and dynamic range remain a challenge. In order to improve the key characteristicsselectivitycharacteristics and of dynamicof the the glucose glucose range remainsensor, sensor, a semipermeable challenge. semipermeable In order membranes membranes to improve were were the emp key employed characteristicsloyed to to coat coat ofthese these the characteristics of the glucose sensor, semipermeable membranes were employed to coat these electrodes.glucoseelectrodes. sensor, semipermeable membranes were employed to coat these electrodes. electrodes. LiLiLi et et et al. al. al. developeddeveloped developed a a anon non-enzymaticnon-enzymatic-enzymatic glucose glucose glucose biosensor biosensor biosensor based based based on on on glassy glassy glassy carbon carbon carbon electrode electrode electrode modified modified modified Li et al. developed a non-enzymatic glucose biosensor based on glassy carbon electrode modified withwithwith hollow hollow hollow nanoparticle nanoparticle nanoparticle (NP) (NP) (NP) chains chains chains of of of platinum platinum platinum on on on porous porous porous gold gold gold nanopart nanoparticles nanoparticlesicles in in in a a a chitosan chitosan chitosan with hollow nanoparticle (NP) chains of platinum on porous gold nanoparticles in a chitosan membranemembranemembrane [35] [35 [35]].. . These These These porous porous porous membranes membranes membranes exhibited exhibited exhibited a aahighly highlyhighly stable stable stable and and and large large large surface surface surface area area area for for for membrane [35]. These porous membranes exhibited a highly stable and large surface area for entrapmententrapmententrapment of of of platinum platinum platinum hollow hollow hollow nanoparticles. nanoparticles. nanoparticles. A AA linear linear linear dynamic dynamic dynamic range range range from from from 3 3 3to toto 7.7 7.77.7 mM mM mM with with with a a a entrapment of platinum hollow nanoparticles. A linear dynamic range from 3 to 7.7 mM with a detectiondetectiondetection limit limit ofof of 11 µ1µMM µM was was was observed. observed. observed. Liu Liu Liu et et al. et al. demonstratedal. demo demonstratednstrated a glassy a aglassy glassy carbon carbon carbon electrode electrode electrode modified modified modified with detection limit of 1 µM was observed. Liu et al. demonstrated a glassy carbon electrode modified withNiOwith hollow NiO NiO hollow sphereshollow spheres in spheres the presence in in the the ofpresence presence chitosan of [ of36 chitosan]. chitosan Fast response [36] [36]. . Fast time,Fast response responsean essential time, time, characteristic an an essential essential of with NiO hollow spheres in the presence of chitosan [36]. Fast response time, an essential characteristicacharacteristic glucose biosensor, of of a aglucose glucose was asbiosensor, biosensor, achieved was bywas thisas as achieved systemachieved (lessby by this thanthis system system 3 s). A(less (less low than detectionthan 3 3s ).s ).A limitA low low detection of detection 0.3 µM characteristic of a glucose biosensor, was as achieved by this system (less than 3 s). A low detection limitatlimit a signalof of 0.3 0.3 toµM µM noise at at a ratioasignal signal (SNR) to to noise noise of 3 ratio was ratio calculated.(SNR) (SNR) of of 3 The3was was chitosancalculated. calculated. membrane The The chitosan chitosan was employedmembrane membrane as was thewas limit of 0.3 µM at a signal to noise ratio (SNR) of 3 was calculated. The chitosan membrane was employedsemipermeableemployed as as the the membrane semipermeable semipermeable to selectively membrane membrane detect to to selectively glucose selectively in thedetect detect presence glucose glucose of in other in the the interfering presence presence of speciesof other other employed as the semipermeable membrane to selectively detect glucose in the presence of other interferinginterfering species species such such as as u ricuric acid acid (UA), (UA), ascorbic ascorbic acid acid (AA) (AA) and and dopamine. dopamine. A A huge huge improvement improvement interfering species such as uric acid (UA), ascorbic acid (AA) and dopamine. A huge improvement fromfrom previous previous work work was was demonstrated demonstrated in in the the sensitivity sensitivity of of the the non non-enzymatic-enzymatic glucose glucose biosensor biosensor by by from previous work was demonstrated in the sensitivity of the non-enzymatic glucose biosensor by WangWang et et al. al., wherein, wherein a notheranother glassy glassy carbon carbon electrode electrode was was modified modified by by electrodepositing electrodepositing dendritic dendritic Wang et al., wherein another glassy carbon electrode was modified by electrodepositing dendritic coppercopper–cobalt–cobalt nanostructures nanostructures (Cu (Cu–Co–Co NSs) NSs) followed followed by by surface surface modification modification by by a areduced reduced g raphenegraphene copper–cobalt nanostructures (Cu–Co NSs) followed by surface modification by a reduced graphene oxideoxide–c–hitosanchitosan nanocomposite nanocomposite [37] [37]. Such. Such a asensor sensor display displayeded a adynamic dynamic range range from from 0.015 0.015 to to 6.95 6.95 mM mM oxide–chitosan nanocomposite [37]. Such a sensor displayed a dynamic range from 0.015 to 6.95 mM

Membranes 2016, 6, 55 7 of 20 such as uric acid (UA), ascorbic acid (AA) and dopamine. A huge improvement from previous work was demonstrated in the sensitivity of the non-enzymatic glucose biosensor by Wang et al., wherein another glassy carbon electrode was modified by electrodepositing dendritic copper–cobalt nanostructures (Cu–Co NSs) followed by surface modification by a reduced graphene oxide–chitosan nanocomposite [37]. Such a sensor displayed a dynamic range from 0.015 to 6.95 mM with a sensitivity of 1.921 mA/cm2·mM and a detection limit of 10 µM[37]. Shen et al. employed a bi-metallic compound of Pd-Au clusters coated with multiple layers of chitosan [38]. The multiple chitosan layers prevented the Pd-Au cluster from leaching out, thereby improving the stability of glucose sensor. Recently, a novel one step co-deposition of nanocomposites of nickel nanoparticle–attapulgite-reduced graphene oxide (Ni NPs/ATP/RGO) on glassy carbon electrodes was demonstrated by Shen et al. [39]. The sensor was designed to detect lower glucose concentration and exhibited a dynamic range from 1 µM to 710 µM and a detection limit of 0.37 µM[39]. A relatively high sensitivity of 1.4144 mA/mM·cm2 and high selectivity amidst interfering species such as UA, AA and 4-Aminophenol was demonstrated. As a result, chitosan membrane-based glucose sensors have gained a tremendous amount of attention due to their advantages in improving the sensitivity and selectivity characteristics of biosensors, as well as being biocompatible and biodegradable. Apart from chitosan, polymer-based semipermeable membranes are commonly employed in the design of biosensors and significant research has been conducted on exploiting their properties to fabricate a novel optimal glucose biosensor. One such work was conducted by Becerik et al., in which bimetals Pt-Pd layered with a conductive polymer and polypyrrole film [40] were employed. It was observed that polypyrrole film porosity improved the surface area showing higher activity towards glucose oxidation compared to bi-metal electrodes without the conductive polymer film. Jiang et al. reported on the fabrication of a glucose biosensor utilizing electrochemical deposition of Ni(OH)2 onto carbon nanotube/polyimide (PI/CNT) membrane [41]. The fabricated sensor exhibited a sensitivity of 2.071 mA/mM·cm2 along with a detection limit of 0.36 µM at SNR of 3. The electrodeposition of Ni(OH)2 onto carbon nanotube/polyimide (PI/CNT) membrane exhibited long-term stability and good reproducibility when operating in human serum and clearly resulted in an improvement in sensitivity compared to the chitosan membrane employed [41]. Conductive polymers, such as polyaniline (PANI) are also being employed as semipermeable membranes to screen against the interfering species such as uric acid, ascorbic acid and acetaminophen. Ahammad et al. employed gold nanoparticles adsorbed onto glassy carbon electrode modified with PANI [42] as the semipermeable membrane. Using electrochemical impedance spectroscopy technique, they were able to detect glucose concentrations from 0.3 mM to 10 mM with a detection limit of 0.1 mM. And indeed, good selectivity towards interfering species uric acid, ascorbic acid and acetaminophen was demonstrated. Another non-enzymatic glucose sensor was fabricated using a core-shell structure of NiCo2O4@Polyaniline nanocomposite via a facile hydrothermal treatment followed by polyaniline coating [43]. Polyaniline membrane is highly conductive, thereby, resulting in a higher electrocatalytic activity compared to NiCo2O4 nanoparticles. The sensor exhibited a linear range up to 4.735 mM with a sensitivity of 4.55 mA/cm2·mM and a detection limit of 0.3833 µM. While these non-enzymatic glucose biosensors with semipermeable membranes exhibit good biosensing characteristics, the limitations of these biosensors, especially for bio-implantable applications, has shifted the research focus towards enzymatic glucose biosensors that overcome the current limitations of non-enzymatic glucose biosensors.

5. Enzymatic Glucose Biosensor Membranes Enzyme-based glucose biosensors employ naturally occurring enzymes derived from living organisms as catalysts to oxidize glucose. They are low-cost, easily renewable and a clean source of catalysts thus, overcoming some of the drawbacks of non-enzymatic glucose biosensors. However, enzymes are very fragile and easily affected by external conditions such as temperature, pH, pressure and humidity [44]. Efforts are underway to stabilize these enzymes once immobilized on an electrode Membranes 2016, 6, 55 8 of 20 substrate to improve their lifetime and stability. To improve the overall performance of an enzymatic glucose biosensor various optimization techniques have been explored [45]. One of the technique is to coat the enzyme with a semipermeable membrane. This membrane prevents the enzyme from leeching out thus, improving the device stability. Also, due to various pore sizes of these membranes, various interferingMembranes 2016 chemical, 6, 55 species are segregated thus, improving the selectivity of the glucose biosensor.8 of 20

5.1.5.1. Cellulose Acetate-BasedAcetate-Based Membranes EarlyEarly researchresearch onon membrane-basedmembrane-based enzymaticenzymatic glucoseglucose biosensorsbiosensors waswas mostlymostly conductedconducted withwith collagencollagen membranes.membranes. Although collagen films films are highly stable and active membranes,membranes, they werewere foundfound to to be be too too thick thick and and fragile fragile [ [46]46].. These These membranes membranes were extensively extensively explored in in the the 1970s 1970s becausebecause of thei theirr high high perm perm-selectivity-selectivity towards towards anions anions and and the the fact fact that that they they can can be bedeposited deposited by byemploying employing film film casting casting or dip or coating dip coating method method [47]. Significant [47]. Significant work has work been hasconducted been conducted by Sternberg by Sternberget al. in which et al. in they which developed they developed various various methods methods for immobilizing for immobilizing glucose glucose oxidase oxidase enzyme enzyme on on a acellu celluloselose acetate acetate membrane membrane [48] [48.]. Their Their work work focused focused on on the the production production of of thin thin and and stable stable membranes. membranes. TheThe optimaloptimal methodmethod involvedinvolved covalentcovalent couplingcoupling ofof bovinebovine serumserum albuminalbumin (BSA)(BSA) to cellulosecellulose acetate membranemembrane andand subsequently subsequently with with the the GOx GOx which which was was then then activated activated with with p-benzoquinone. p-benzoquinone. Such workSuch yieldedwork yielded thin membranes thin membranes of 5–20 ofµ 5m–20 with µm high with surface high surface activities activities of 1–3 U/cmof 1–33 U/cm³and stability and stability of up toof 3up months. to 3 months. Recently, Recently, the cellulose the acetate cellulose membrane acetate hasmembrane resurfaced has as resurfaced a semipermeable as a semipermeable membrane for enzymaticmembrane glucose for enzym biosensors.atic glucose Glucose biosensors. biosensors Glucose consisting biosensor of biologicals consisting and electronic of biological water-based and inkelectronic containing water GOx-based and conducting ink containing polymer GOx blend and poly(3,4-ethylenedioxythiophene/polystyrene conducting polymer blend poly(3,4- sulphonicethylenedioxythiophene/polystyrene acid (PEDOT/PSS) was thermally sulphonic inkjet acid printed (PEDOT/PSS) on an indium was tin thermally oxide (ITO) inkjet glass printed substrate on byan Setti indium et al.tin [ 49 oxide]. This (ITO) device glass was substrate encapsulated by Setti in et a cellulose al. [49]. This acetate device semipermeable was encapsulated membrane in a viacellulose dip-coating. acetate semipermeable The glucose biosensor membrane produced via dip a-coating. linear response The glucose up tobiosensor 60 mM withproduced a sensitivity a linear ofresponse 0.00643 up mA/mM to 60 mM·cm 2with. Figure a sensitivity5 illustrates of the0.00643 two-layer mA/mM· cellulosecm2. Figure acetate 5 membraneillustrates the employed two-layer in thecellulose development acetate membrane of an electrochemical employed in measurement the development set up of comprising an electrochemical of a glucose measurement biosensor set and up a complementarycomprising of ametal glucose oxide biosensor semiconductor and a (CMOS) complementary potentiostat metal [50]. oxide Although semiconductor the sensitivity (CMOS of the) biosensorpotentiostat was [50] relatively. Although low, the membranesensitivity wasof the capable biosensor of eliminating was relatively ascorbic low, acid, theL-glutathione membrane was and Lcapable-cysteine, of impedingeliminating diffusion ascorbic through acid, L the-glutathione membrane and to the L-cysteine biorecognition, impeding element, diffusion the GOx-modified through the bioelectrodemembrane to as the shown biorecognition in Figure5 .element, the GOx-modified bioelectrode as shown in Figure 5.

FigureFigure 5.5. Electrochemical measurement setup for glucose sensing, comprising multi-layermulti-layer membrane andand CMOSCMOS potentiostat.potentiostat.

5.2.5.2. Nafion-BasedNafion-Based MembranesMembranes PerfluorosulphonicPerfluorosulphonic acid polymer, commonly known as nafionnafion is one of the the mostmost commonly commonly employedemployed semipermeable semipermeable membrane in the the design design and and development development of of glucose glucose biosensors. biosensors. ThisThis perfluorinatedperfluorinated cation-exchangecation-exchange polymer polymer with with a hydrophobic a hydrophobic perfluoro perfluoro backbone backbone and pendant and pendant sulfonic sulfonic acid groups allows for the permeation of hydrogen peroxide while restricting the passage of anions (e.g., ascorbic acid and uric acid) across the membrane [51]. This thereby reduces electrode fouling and interference by ascorbic acid (as shown in Figure 6) as a result of the negatively charged pendant sulfonic acid groups that prohibit the passage of these negatively charged analytes.

Membranes 2016, 6, 55 9 of 20 acid groups allows for the permeation of hydrogen peroxide while restricting the passage of anions (e.g., ascorbic acid and uric acid) across the membrane [51]. This thereby reduces electrode fouling and interference by ascorbic acid (as shown in Figure6) as a result of the negatively charged pendant Membranessulfonic acid2016, groups6, 55 that prohibit the passage of these negatively charged analytes. 9 of 20

Figure 6. Schematic representation of nafion membrane restricting passage of interfering analyte, Figure 6. Schematic representation of nafion membrane restricting passage of interfering analyte, ascorbic acid. ascorbic acid.

Harrison et al. modified a Pt electrode with GOx followed by nafion coating [52]. The thickness of theHarrison nafion membrane et al. modified was a1.7 Pt µm electrode thick to with enable GOx continuous followed by innafion vitro measurement coating [52]. Theof glucose thickness in of the nafion membrane was 1.7 µm thick to enable continuous in vitro measurement of glucose in blood at 37 °C.◦ A linear response of up to 28 mM with a response time ranging from 5 to 17 s was observedblood at 37whichC. Awas linear significantly response higher of up than to 28 bioelectrodes mM with a response coated with time cellulose ranging acetate from 5 membranes. to 17 s was Aobserved new mixed which membrane was significantly material higherconsisting than of bioelectrodes nafion and laponite coated withgel was cellulose used to acetate encapsulate membranes. GOx Ain neworder mixed to modulate membrane the enzyme material loading consisting in the of nafionbiomembrane. and laponite The sensitivity gel was used of glucose to encapsulate sensing GOxwas foundin order to tobe modulate directly proportional the enzyme loadingto the enzyme in the biomembrane.content in the gel The membrane. sensitivity A of GOx glucose to laponite sensing ratio was offound 3.3 achieved to be directly a sensitivity proportional of 132 to mA/ themM· enzymecm2 over content a linear in the dynamic gel membrane. range from A GOx 0.01 to mM laponite to 20 mM. ratio · 2 Theof 3.3 effect achieved of interfering a sensitivity species of 132 such mA/mM as ascorbate,cm over urate a linear and acetaminophen dynamic range was from reduced 0.01 mM by to a 20 factor mM. Theof 4 with effect the of interferinguse of nafion species membra suchne as and ascorbate, polyphenol urate oxidase and acetaminophen [53]. was reduced by a factor of 4 withCarbon the nanotubes use of nafion have membrane been explored and polyphenol as electrode oxidase substrates [53]. for their advantages over other metallicCarbon and nanotubesglassy carbon have electrodes. been explored Some asof electrodethe advantages substrates include for strong their advantages bonding between over other the atomsmetallic and and the glassy tubes carbonand extreme electrodes. aspect Some ratios of thus, the advantagesimproving the include conductivity strong bonding and surface between area thefor enzymeatoms and immobilization. the tubes and Current extreme research aspect ratios has demonstrated thus, improving that the GOx conductivity and palladium and surface nanoparticles area for canenzyme be readily immobilization. co-deposit Currented on a researchnafion-solubilized has demonstrated carbon thatnanotube GOx and[54]. palladium The fabricated nanoparticles glucose biosensorcan be readily showed co-deposited a linear response on a nafion-solubilized of up to 12 mM with carbon a detection nanotube limit [54 of]. 0.15 The mM fabricated with a glucoseSNR of 3.biosensor The nafion showed coating a linear eliminated response the of up effects to 12 of mM common with a detection interfering limit species of 0.15 such mM as with uric a acidSNR andof 3. Theascorbic nafion acid. coating The eliminateduse of carbon the nanotubes effects of common as the electrode interfering substrates species such for asenzyme uric acid immobilization and ascorbic wasacid. very The use effective of carbon and nanotubes resulted in as the electrode exploration substrates of carbon for nanowiresenzyme immobilization and a more was com verypact aggregatedeffective and chain resulted of multi in the-wall explorationed carbon of carbon nanotubes nanowires (i.e., Buckypaper) and a more compact as the substrates aggregated for chain high of- densitymulti-walled enzyme carbon loading. nanotubes (i.e., Buckypaper) as the substrates for high-density enzyme loading. A self-powered self-powered glucose glucose biosensing biosensing microsystem microsystem was was recently recently fabricated fabricated by Slaughter by Slaughter et al., whichet al., waswhich powered was powered by a biofuel by a biofuel cell consisting cell consisting of pyroloquinoline of pyroloquinoline quinone quinone glucose dehydrogenase-modifiedglucose dehydrogenase- modifiedbioanode bioanode and laccase-modified and laccase-modified biocathode biocathode [6]. A transducer [6]. A transducer element element capacitor capacitor connected connected at the atoutput the output of charge of charge pump pump was used was toused sense to sense glucose glucose via monitoring via monitoring the charge the charge cycle cycle across across it. The it. Thebioelectrodes bioelectrodes were were coated coated with with nafion nafion membrane membrane which which prevented prevented the enzymesthe enzymes from from leeching leeching out outand andeffectively effectively screening screening against against competing competing analytes. analytes. Such novel Such system novel exhibited system exhibited a stable operation a stable operationover 97 days overin 97 vitro days. This in vitro novel. This biosensing novel biosensing system showed system showed promise promise in effectively in effectively screening screen againsting againstinterfering interfering analytes analytes because itbecause did not it produce did not theproduce necessary the necessary 700 mV required 700 mV to required break down to break interfering down interferinganalytes [55 analytes]. The presence [55]. The of presence interfering of interfering analytes has analytes no impact has no in impact the glucose in the readings glucose andreadings thus, andresults thus, in anresult improvements in an improvement in selectivity. in selectivity. In addition, aa glucoseglucose biosensorbiosensor waswas developed developed to to measure measure the the cerebral cerebral glucose glucose levels levels in in order order to tounderstand understand the mechanisms the mechanism involvings involving insulin and insulin anti-hypertensive and anti-hypertensive drugs regulated drug ins hyperglycemicregulated in hyperdiabeticglycemic rats [56 diabetic]. This custom-builtrats [56]. This glucose custom micro-biosensor-built glucose micro was-biosensor implanted was in theimplanted striatum. in Thethe striatum. The biosensor comprised of GOx trapped inside a poly 4-vinylpyridine (P4VP) membrane and deposited on Pt electrode. Furthermore, the biosensor was coated with a thin nafion layer. It was observed that the administration of insulin had no significant effect on both hyperglycemic and diabetic rats but the anti-hypertensive drug lowered the glucose levels in the brain. This was the first implementation of a glucose biosensor developed to measure the cerebral glucose levels and thus, shows promise to better understand the complex mechanism of the brain.

Membranes 2016, 6, 55 10 of 20 biosensor comprised of GOx trapped inside a poly 4-vinylpyridine (P4VP) membrane and deposited on Pt electrode. Furthermore, the biosensor was coated with a thin nafion layer. It was observed that the administration of insulin had no significant effect on both hyperglycemic and diabetic rats but the anti-hypertensive drug lowered the glucose levels in the brain. This was the first implementation ofMembranes a glucose 2016 biosensor, 6, 55 developed to measure the cerebral glucose levels and thus, shows promise10 of to20 better understand the complex mechanism of the brain. Besides showing showing great great promise promise in in screening screening against against negatively negatively charged charged analytes, analytes, nafion nafion has been has beenshown shown to be to in beeffective ineffective with with neutrally neutrally charged charged analytes, analytes, such such as as acetaminophen. acetaminophen. To To prohibit acetaminophen passage, passage, composite composite cellulose cellulose acetate acetate and and nafion nafion membrane membraness have have been been designed designed to toeliminate eliminate the theof passage of passage acetaminophen acetaminophen at the atcost the of costpoor ofsensitivity poor sensitivity [57]. However [57]., However,such composite such compositemembranes membranes can be useful can inbe differentuseful in glucosedifferent biosensors glucose biosensors,, except for except the first for- thegeneration first-generation glucose glucosebiosensors. biosensors.

5.3.5.3. Other Polymer-BasedPolymer-Based Membranes Due to to the the redox-switchable redox-switchable nature nature of polypyrrole, of polypyrrole, it was exploited it was exploited in the design in and the development design and ofdevelopment glucose biosensor of glucose [58 ],biosensor wherein [58] a glucose, wherein biosensor a glucose was biosensor designed was by Ramanaviˇciusetdesigned by Ramanavičius al. In this study,et al. In glucosethis study oxidase, glucose nanoparticles oxidase nanoparticles were encapsulated were encapsulated within within a polypyrrole a polypyrrole membrane membrane [58]. The[58]. incorporation The incorporation of polypyrrole, of polypyrrole, ahighly a highly conductive conductive polymer, polymer, was was shown shown to to increase increase the M cat Michalis-MentenMichalis-Menten constantconstant (K(KM) and the rate of reaction (K(Kcat).). An An alternative alternative approach approach was employed to electropolymerizedelectropolymerized m-phenylenem-phenylene diamine diamine film film with with GOx, GOx, lactate lactate oxidase oxidase and and glutamate glutamate oxidase oxidase on aon carbon a carbon fiber fiber electrode electrod coverede covered with electrometalized with electrometalized ruthenium ruthenium layer [59 layer]. This [59] biosensor. This biosensor exhibited aexhibited relatively a smallrelatively dynamic small range dynamic of up range to 4 mMof up for to glucose4 mM for with glucose a detection with a limitdetection of 0.5 limitµM withof 0.5 SNR µM ofwith 3. ItSNR was of operationally 3. It was operationally stable for overstable 10 for h inover a dynamic 10 h in a environmentdynamic environment at 36 ◦C and at 36 pH °C of and 7.4. pH Such of a7.4. system Such alloweda system for allowed characterization for characterization of the glucose of thein glucose vivo, however, in vivo, ithowever, was incapable it was incapable of detecting of normaldetecting glucose normal levels glucose as well levels as hyperglycemiaas well as hyperglycemia due to its very due narrow to its very dynamic narrow range. dynamic Additionally, range. compositeAdditionally, polymer composite layers polymer such as layer polyurethanes such as polyurethane and nafion continue and nafion to gain continue attention to gain in completeattention screeningin complete against screening interfering against species interfering such as species ascorbic such acid, as uric ascorbic acid, acid,L-cysteine, uric acid, acetaminophen, L-cysteine, dopamine,acetaminophen, aspartic dopamine, acid, glutamine aspartic and acid, homovanillic glutamine acid and [59 homovanillic]. A co-polymer acid hydrogel [59]. A consisting co-polymer of 1,3-diaminobenzenehydrogel consisting hasof 1, been3-diaminobenzene used in the design has ofbeen a novel used biosensor in the design array of capable a novel of biosensor simultaneously array sensingcapable glucose,of simultaneously lactate, glutamine sensing glucose, and glutamate lactate, is glutamine shown in and Figure glutamate7. The system is shown comprised in Figure of a7. glassThe system chip with comprised integrated of a biosensorglass chip arraywith integrated and a gold biosensor electrode array to provide and a electricalgold electrode continuity. to provide This biosensorelectrical continuity. system operated This biosensor over a wide system dynamic operated range over of a 0.1 wide mM–35 dynamic mM. range However, of 0.1 the mM sensitivity–35 mM. ofHowever, the biosensor the sensitivity was very of low the (5–20 biosensor nA/mM was cm very2). Thelow biosensor(5–20 nA/mM system cm exhibited2). The biosensor an operational system stabilityexhibited of an a littleoperational over 4 weeksstability and of a storagelittle over stability 4 weeks of and 2 years a storage with less stability than 0.5of 2 mM years in responsewith less tothan interference. 0.5 mM in response to interference.

Figure 7. The biosensor array with glucose, lactate, glutamine, and glutamate sensor [[60]60]..

A glucose biosensor, in which glucose oxidase enzyme immobilized on Pt electrode and A glucose biosensor, in which glucose oxidase enzyme immobilized on Pt electrode and subsequently coated with a permselective membrane poly(4-vinylpyridine-co-styrene) [61] has been subsequently coated with a permselective membrane poly(4-vinylpyridine-co-styrene) [61] has been demonstrated with dynamic range in the lower glucose concentration range (0.01 mM–1.5 mM) that exhibited a high sensitivity of 30 mA/mM·cm2. The membrane employed was found to be successful in the elimination of the effects of ascorbic acid, urate and p-acetaminophen. In order to increase the sensitivity of the glucose biosensor, GOx was immobilized in the presence of BSA on a nano-yarn carbon nanotube followed by coating with epoxy-polyurethane. A 7.5-fold increase in the glucose sensitivity was observed compared to the use of Pt–Ir coil-based electrode and exhibited an operating stability over 70 days. Polyphenol-polyurethane electropolymerization techniques as shown in Figure 8 have been employed with xerogel to encapsulate GOx [62]. The system achieved a linear

Membranes 2016, 6, 55 11 of 20 demonstrated with dynamic range in the lower glucose concentration range (0.01 mM–1.5 mM) that exhibited a high sensitivity of 30 mA/mM·cm2. The membrane employed was found to be successful in the elimination of the effects of ascorbic acid, urate and p-acetaminophen. In order to increase the sensitivity of the glucose biosensor, GOx was immobilized in the presence of BSA on a nano-yarn carbon nanotube followed by coating with epoxy-polyurethane. A 7.5-fold increase in the glucose sensitivity was observed compared to the use of Pt–Ir coil-based electrode and exhibited an operating stabilityMembranes over 2016, 706, 55 days. Polyphenol-polyurethane electropolymerization techniques as shown in Figure11 of 208 have been employed with xerogel to encapsulate GOx [62]. The system achieved a linear dynamic rangedynamic over range 28 mM over with 28 mM a very with fast a responsevery fast time.response The membranetime. The membrane achieved selective achieved glucose selective sensing glucose in thesensing presence in the of presence acetaminophen, of acetaminophen, ascorbic acid, ascorbic sodium acid, nitrate, sodium oxalic nitrate, acid andoxalic uric acid acid. and Clearly, uric acid. the incorporationClearly, the incorporation of various polymer-based of various polymer membranes-based have membranes been shown have to been be critical shown in to improving be critical the in biosensor’simproving the selectivity, biosenso sensitivityr’s selectivity, and linearsensitivity dynamic and linear range. dynamic range.

Figure 8. 8. Schematic diagram of a xerogel-based, xerogel-based, first-generationfirst-generation amperometric glucose biosensor biosensor featuringfeaturing an enzymeenzyme dopeddoped andand diffusion-limitingdiffusion-limiting xerogelxerogel layerslayers andand cappedcapped withwith semipermeablesemipermeable electropolymerized polyphenolpolyphenol andand polyurethanepolyurethane outerouter membranes.membranes.

5.4.5.4. Chitosan-BasedChitosan-Based Membrane Chitin, a naturally occurring chemical,chemical, is abundantly available in crustaceans and is known to consist ofof 2-acetamido-2-deoxy-2-acetamido-2-deoxy-β-D--glucose.glucose. Its Its immunogenicity immunogenicity is is ex exceptionallyceptionally low low,, along with it being a highlyhighly insolubleinsoluble material.material. Chitosan is the N-deacetylated-deacetylated derivative of chitin and is highly biocompatible. As As a a result, result, it has it has become become a prominent a prominent semipermeable semipermeable membrane membrane in enzymatic in enzymatic glucose sensors.glucose One sensor ofs the. One prominent of the works promin thatent improved works that the improvedelectron transfer the electron rate was transfer demonstrated rate was by Liudemonstrated et al., wherein by Liu GOx et was al., trappedwherein in GOx a composite was trapped mixture in ofa composite carbon nanotube mixture and of chitosancarbon nanotube resulting inand a combinationchitosan resulting that enabled in a combination the enhancement that enabled in the the direct enhancement electron transfer in the rate direct (7.73/s) electron which transfer was morerate (7.73/s) than one-fold which was increase more than over one GOx-adsorbed-fold increase on over carbon GOx nanotubes-adsorbed on (3.10/s) carbon [63 nanotubes]. Moreover, (3.10/s) the sensitivity[63]. Moreover, of such the biosensor sensitivity was of calculatedsuch biosensor to be was 0.577 calculated mA/mM ·tocm be2. The0.577 use mA/ ofmM· chitosancm2. membraneThe use of ensuredchitosan that membrane enzymes ensured stay entrapped, that enzymes thus improving stay entrapped the stability, thus impr of theoving biosensor. the stability Since of metal the surfacesbiosensor. also Since possess metal high surfaces affinity also towards possess enzyme high affinity immobilization, towards enzyme Zeng et immobilization, al. immobilized GOxZengon et palladiumal. immobilized nanoparticles GOx on modified palladium with chitosan nanoparticles membrane modified [64]. Improvement with chitosan in biocompatibilitymembrane [64]. andImprovement hydrophilicity in biocompatibility was observed with and ahydrophili low reactioncity rate was constant observed ensuring with a low enhanced reaction enzyme rate constant affinity toensuring glucose. enhanced The sensor enzyme exhibited affinity a to linear glucose. dynamic The sensor range exhibited from 1 µ aM linear to 1 dynamic mM with range a detection from 1 µM to 1 mM with a detection limit of 0.2 µM at SNR of 3 and a sensitivity of 0.031 mA/mM·cm2. Ang et al. recently developed a glucose biosensor and characterized it in a fruit [65]. The glucose biosensor was constructed from a Pt electrode modified with glucose oxidase immobilized in a chitosan membrane. There was an observed improvement in the rate of reaction when compared to the system developed by Zeng et al. The limit of detection for this biosensor was observed to be 0.05 mM at SNR of 3. The sensor showed good stability with high enzyme retention activity. It also showed good repeatability and reproducibility with a relative standard deviation of 2.30% and 3.70% in the collected data, respectively. Another application-based glucose biosensor was recently developed, in which Prussian blue modified graphene strings were immobilized with GOx within a biocompatible chitosan layer. A linear dynamic range from 0.01 mM to 1 mM with a response time of less than 3 s

Membranes 2016, 6, 55 12 of 20 limit of 0.2 µM at SNR of 3 and a sensitivity of 0.031 mA/mM·cm2. Ang et al. recently developed a glucose biosensor and characterized it in a fruit [65]. The glucose biosensor was constructed from a Pt electrode modified with glucose oxidase immobilized in a chitosan membrane. There was an observed improvement in the rate of reaction when compared to the system developed by Zeng et al. The limit of detection for this biosensor was observed to be 0.05 mM at SNR of 3. The sensor showed good stability with high enzyme retention activity. It also showed good repeatability and reproducibility with a relative standard deviation of 2.30% and 3.70% in the collected data, respectively. Another application-based glucose biosensor was recently developed, in which Prussian blue modified graphene strings were immobilized with GOx within a biocompatible chitosan layer. A linear dynamic range from 0.01 mM to 1 mM with a response time of less than 3 s was reported [66]. Sensitivity of glucose biosensor was observed to be 0.641 µA/mM·cm2. However, this combination resulted in a decrease in the chitosan membrane to completely eliminate or screen against ascorbic acid, uric acid, galactose and acetaminophen.

5.5. Poly(2-hydroxyethyl Methacrylate) (pHEMA)-Based Membranes Flexible and hydrophilic hydrogels have also been investigated extensively as semipermeable membranes in enzymatic glucose biosensors [67,68]. Hydrogel membranes are made up of mostly water and have been found to be biocompatible. They are commonly used to entrap enzymes in the development of glucose biosensors. A glucose-permeable hydrogel made from crosslinking 8-armed amine terminated poly(ethylene glycol) (PEG) in aqueous solution at room temperature was assayed for biocompatibility in a rat model [69]. Although it was observed that the presence of the cross-linked PEG hydrogel deteriorated and resulted in 34% drop in the biosensor sensitivity when characterized in glucose concentration from 0 to 30 mM, these gels were found to be good candidates for bio-implantable biosensors. Arica et al. characterized the effects of various parameters such as temperature, concentration of hydrogel components and storage life of poly(2-hydroxyethyl methacrylate) (pHEMA)-based hydrogels as semipermeable membranes [70]. GOx was entrapped in the pHEMA membrane through matrix entrapment. It was observed that the affinity of GOx towards glucose decreased substantially. Although membranes with the highest enzyme activity were found to be most permeable, thereby increasing the enzyme content of the membrane adversely affected the biosensor’s activity. The membrane permeability was however, observed to increase at low pHEMA concentrations. Noting the importance of pHEMA gels, glucose biosensors were fabricated using pHEMA, poly(ethylene glycol) and tetra-acrylate and ethylene dimethacrylate [71–75]. Quinn et al. electrically wired GOx to a gold current collector via a redox polymer, which resulted in a 45% ± 28% decrease in the biosensor response at physiological condition [71]. Brahim et al. developed an ‘intelligent’ hydrogel by incorporating polypyrrole (PPy) within the highly pHEMA-based hydrogel to yield a PPy-pHEMA hydrogel [75]. It was observed that the ‘intelligent’ hydrogel retains its hydration and as well as the electroactivity of the conducting polymer, polypyrrole. This PPy-pHEMA hydrogel composite in Figure9 was employed as a semipermeable membrane in the construction of a dual glucose and lactate biosensors by Guiseppi-Elie et al. [74]. The PPy component provided interference screening capabilities, whereas the pHEMA provided excellent in vivo biocompatibility. A linear dynamic range of 0.10–13.0 mM for glucose and up to 90 mM for lactate was observed. A PEDOT component was used in place of the polypyrrole component to create p(HEMA)-PEDOT membrane that enhanced the stability of the biosensor [73]. The biosensor exhibited stability over 90 days and selectively screened against the competing analyte fructose. Membranes 2016, 6, 55 13 of 20 Membranes 2016, 6, 55 13 of 20

FigureFigure 9. pHEMApHEMA and and PPy PPy hydrogel hydrogel membrane membrane comprising comprising o off entrapped entrapped oxidase oxidase enzyme enzyme [74] [74].. pHEMA:pHEMA: poly( poly(2-hydroxyethyl2-hydroxyethyl methacrylate) methacrylate);; PPy: polypyrrole polypyrrole..

The PPy component provided interference screening capabilities, whereas the pHEMA provided A needle-shaped platinum enzymatic glucose biosensor based on GOx immobilized in a pHEMA excellent in vivo biocompatibility. A linear dynamic range of 0.10–13.0 mM for glucose and up to 90 membrane that was coated with an outer membrane composed of a pHEMA/polyurethane composite mM for lactate was observed. A PEDOT component was used in place of the polypyrrole component mixture was developed by Shaw et al. [76]. It exhibited long-term stability when operating in 5 mM to create p(HEMA)-PEDOT membrane that enhanced the stability of the biosensor [73]. The biosensor glucose solution in vitro and suffered no significant loss over a 60 h of continuous operation. Moreover, exhibited stability over 90 days and selectively screened against the competing analyte fructose. a linear dynamic range of up to 20 mM glucose was observed. A needle-shaped platinum enzymatic glucose biosensor based on GOx immobilized in a To overcome some of the drawbacks of GOx, glucose dehydrogenase enzymes are being employed pHEMA membrane that was coated with an outer membrane composed of a pHEMA/polyurethane in the development of glucose biosensors. A glucose biosensor was developed by immobilizing composite mixture was developed by Shaw et al. [76]. It exhibited long-term stability when operating glucose dehydrogenase (GDH) and nicotinamide adenine dinucleotide phosphate (NADP+) coenzyme in 5 mM glucose solution in vitro and suffered no significant loss over a 60 h of continuous operation. on a biocomposite made of graphite powder and polymethacrylate [77]. The procedure was Moreover, a linear dynamic range of up to 20 mM glucose was observed. highly reproducible and exhibited stability over 120 days. Table2 summarizes the glucose To overcome some of the drawbacks of GOx, glucose dehydrogenase enzymes are being biosensor employing various semipermeable membranes that have been employed in the design employed in the development of glucose biosensors. A glucose biosensor was developed by and development of glucose biosensor membranes. immobilizing glucose dehydrogenase (GDH) and nicotinamide adenine dinucleotide phosphate (NADP+) coenzyme on a biocomposite made of graphite powder and polymethacrylate [77]. The procedure was highly reproducible and exhibited stability over 120 days. Table 2 summarizes the glucose biosensor employing various semipermeable membranes that have been employed in the design and development of glucose biosensor membranes.

6. Conclusions Semipermeable membranes play a significant role in improving glucose sensor characteristics. Cellulose acetate membranes were one of the earliest and most commonly used commercial form of reverse osmosis membranes. These membranes have an added benefit of low cost and high tolerance towards chlorine, which is essential since they are more susceptible to biodegradation. They quickly gained significant attention as semipermeable membranes for glucose biosensors. They were

Membranes 2016, 6, 55 14 of 20

Table 2. Summary of Semipermeable Membrane in Glucose Biosensors.

Linear Dynamic Reference Substrate Enzyme Analyte Membrane Sensitivity (µA/mM·cm2) Author Range (mM) Number Biological- and Glucose oxidase Glucose Cellulose acetate 6.43 (µA/M·cm2) Upto 60 mM Setti, L., et al. (2005) [49] water-based inks Polydimethylsiloxane, Ceramic Glucose oxidase Glucose 0.1922 (µA/mM·cm2) Upto 200 mM Mross, Stefan, et al. (2015) [50] Cellulose acetate Platinum Glucose oxidase Glucose Nafion 176.18 (µA/mM·cm2) Upto 28 mM Harrison, D., et al. (1988) [52] Platinum Glucose oxidase Glucose Nafion 132 (mA/mM·cm2) 0.01–20 mM Poyard, S., et al. (1998) [53] Carbon nanotube Glucose oxidase Glucose Nafion – Upto 12 mM Lim, San Hua, et al. (2005) [54] Glucose oxidase + Glucose, Glucose (upto 4 mM), Carbon fiber + Lactate oxidase + Glutamate and m-phenylene diamine – Glutamate (upto 0.25 mM), Schuvailo, O.M., et al. (2006) [59] Ruthenium Glutamate oxidase Lactate Lactate (upto 1.75 mM) Glucose (5–20 (nA/mM·mm2)), Lactate Glucose, Glucose (0.1–35 mM), Glucose oxidase + (10–40 (nA/mM·mm2)), Glutamine, Lactate (0.05–15 mM), Platinum Lactate oxidase + 1,3-Diaminobenzene Glutamine (30 Moser, I., et al. (2002) [60] Glutamate and Glutamine (0.05–10 mM), Glutamate oxidase (nA/mM·mm2)), Lactate Glutamate (0.001–5 mM) Glutamate (20–400 (nA/mM·mm2)) Poly(4-vinylpyridine- Platinum Glucose oxidase Glucose 30 (mA/mM·cm2) 0.01–1.5 mM Poyard, S., et al. (1999) [61] co-styrene) Polyphenol + Platinum Glucose oxidase Glucose 354.23 (µA/mM·cm2) ≥24–28 mM Poulos, N.G., et al. (2015) [62] Polyurethane Carbon nanotube Glucose oxidase Glucose Chitosan 184.4 (µA/mM·cm2) 0–7.8 mM Liu, Ying, et al. (2005) [63] Palladium nanoparticles + Glucose oxidase Glucose Chitosan 31.2 (µA/mM·cm2) 0.001–1 mM Zeng, Qiong, et al. (2011) [64] graphene Platinum Glucose oxidase Glucose Chitosan 10.18 (mA/mM·cm2) 0.01–15 mM Ang, L.F., et al. (2015) [59] Prussian blue Glucose oxidase Glucose Chitosan 641.3 (µA/mM·cm2) 0.03–1 mM Lee, Seung Ho, et al. (2016) [60] graphite strings Gold wire Glucose oxidase Glucose Poly(ethylene glycol) (PEG) 616.11 (µA/mM·cm2) 0–30 mM Quinn, C.A., et al. (1997) [63] Membranes 2016, 6, 55 15 of 20

6. Conclusions Semipermeable membranes play a significant role in improving glucose sensor characteristics. Cellulose acetate membranes were one of the earliest and most commonly used commercial form of reverse osmosis membranes. These membranes have an added benefit of low cost and high tolerance towards chlorine, which is essential since they are more susceptible to biodegradation. They quickly gained significant attention as semipermeable membranes for glucose biosensors. They were typically manufactured as thick membranes, which limit analyte diffusion and were not ideal for glucose biosensing. For use as thin membranes with lower permeability, they are subjected to high pressures, which result in an increase in manufacturing cost. Moreover, they are vulnerable to in the presence of acids and alkalies, which render them inferior to salt rejection. Physiological fluids have fair amount of salt content and that can affect the performance of these membranes when employed in glucose biosensing. With a narrow pH range of 4–8 and a temperature range of 0–35 ◦C, these membranes fail to operate at physiological temperatures and hence are now being replaced by other semipermeable membranes that have wide operating pH and temperature ranges. Nafion has been explored by many researchers to entrap enzymes and create a microenvironment to improve the long-term stability of biosensors [78,79]. These membranes overcome the challenges exhibited by cellulose acetate membranes and can operate in wide ranges of pH and temperature (<100 ◦C) [80]. Although this membrane exhibits great advantages, the biggest drawback is its poor performance under low humidification conditions [81], high oxygen permeability (9.3 × 10−12 mol/cm2·s) and its susceptibility to membrane fouling, which in turn limits the operational stability of the biosensors. These limitations make nafion membrane an unpopular choice for commercial biosensors [82]. Due to these observed limitations, the research direction shifted towards exploration of other polymer membranes. The highly conductive (2–100 S/cm) and switchable nature of the polypyrrole membrane along with its high thermal stability result in a higher ionic transfer rate [82] than with nafion membranes. This has resulted in its incorporation into various semipermeable membranes. Polyphenols structures, on the other hand, consist of large π-electron configurations that enable them to have great affinity towards enzymes [83], whereas polyurethane films have been used in combination with other polymer membranes to provide flexible but hard membrane structures to shield the bioelectrode from physical damage. Although different polymeric membranes have been used to minimize the effect of interferents, there is still a need for variety of biocompatible materials that can easily screen against interferents and improve biosensor characteristics if they are to be used in vivo. Overall, nafion and chitosan-based membranes have exhibited high biocompatibility in addition to enhancing the sensitivity and selectivity of biosensors. The added benefit of incorporating chitosan-based films is that they exhibit excellent oxygen barrier ability [84] and are biodegradable, non-toxic, inert and hydrophilic [85]. Non-toxicity along with excellent chemical stability has made PPy-pHEMA membranes a common choice to minimize the effects of interferents in glucose biosensing systems. pHEMA semipermeable membranes like chitosan are biocompatible. Moreover, they possess stronger mechanical properties compared to almost all other polymers [86]. Their mechanical properties can be easily improved with bulk polymerization and copolymerization [87,88]. Although these semipermeable membranes exhibit excellent enzyme retaining ability along with complimentary physical properties that improve the performance of biosensors, very rarely have they been implemented in practice due to the increase in complexity of bioelectrode design. Due to the fragile nature of enzymes, the in situ synthesis of these semipermeable membranes is likely to negatively impact the enzymes, thereby altering their properties and possibly denaturing/deactivating them. In most glucose biosensors, the bioelectrodes are first modified with the biorecognition element within a semipermeable membrane followed by an outer membrane coating that is employed to protect the inner membrane and thus, the biorecognition element. The addition of a second or even a multiple semipermeable membrane layers further limits the passage of the desired analyte and thus, affects the overall performance of the glucose biosensors. Membranes 2016, 6, 55 16 of 20

Although Tipnis et al. demonstrated layer by layer development of multi-layer membrane for glucose biosensing [89], further work needs to be done in optimizing the protocol before it can be use in practice. The various membranes reviewed have been shown to improve the electron transfer rate along with selectively oxidizing glucose in the presence of common interfering species such as ascorbic acid, uric acid and acetaminophen, fructose. Furthermore, the use of biocompatible semipermeable membranes has been shown to increase successful implantation along with good operational stability in vivo. Although there has been a tremendous improvement in sensing characteristics, long-term stability and operational lifetime remain a challenge to surpass the performance of the already available commercial glucose biosensors. Significant work is underway in which microsystem technology offers a promising future in the glucose biosensor industry, potentially replacing present glucose monitoring systems which are bulky, battery-powered and require frequent recalibration.

Acknowledgments: This work was supported by National Science Foundation, United States (Award ECCS-# #1349603). Author Contributions: Gymama Slaughter conceived and designed this study. Tanmay Kulkarni carried out the study and was supervised by Gymama Slaughter. All authors discussed the results, interpreted the data, wrote the manuscript and approved the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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