Research Article Fabrication of Patterned Integrated Electrochemical...

Research ArticleFabrication of Patterned Integrated Electrochemical Sensors

Muhammad Mujeeb-U-Rahman Dvin Adalian and Axel Scherer

California Institute of Technology Department of Physics and Applied Physics Pasadena CA 91125 USA

Correspondence should be addressed to Muhammad Mujeeb-U-Rahman mrahmancaltechedu

Received 18 March 2015 Revised 15 June 2015 Accepted 25 June 2015

Academic Editor Carlos R Cabrera

Copyright copy 2015 Muhammad Mujeeb-U-Rahman et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Fabrication of integrated electrochemical sensors is an important step towards realizing fully integrated and truly wireless platformsfor many local real-time sensing applications Micronanoscale patterning of small area electrochemical sensor surfaces enhancesthe sensor performance to overcome the limitations resulting from their small surface area and thus is the key to the successfulminiaturization of integrated platformsWe have demonstrated the microfabrication of electrochemical sensors utilizing top-downlithography and etching techniques on silicon and CMOS substrates This choice of fabrication avoids the need of bottom-uptechniques that are not compatiblewith establishedmethods for fabricating electronics (eg CMOS)which form the industrial basisofmost integratedmicrosystemsWepresent the results of applyingmicrofabricated sensors to variousmeasurement problems withspecial attention to their use for continuous DNA and glucose sensing Our results demonstrate the advantages of using micro- andnanofabrication techniques for the miniaturization and optimization of modern sensing platforms that employ well-establishedelectronic measurement techniques

1 Introduction

Ultra-small scale integrated electrochemical sensors havegained considerable interest as solutions to diagnostic mon-itoring situations requiring a small footprint One categoryof microsensors that benefits fromminiaturization is medicalimplants which require fully autonomous sensing platformsalong with electronic driving circuitry within the smallestpossible volume [1] Electrochemical sensing technology inparticular is a very attractive solution for health monitoringas it is possible to integrate electrochemical circuits withdevice electronics on a comparatively small size scale [2]Microscale and nanoscale patterning of the sensor surfacesholds the key to making these devices perform effectivelyand to reducing the sensor impedance [3] Such techniquescan also be used to enhance the performance of large scaleelectrodes made via more conventional methods (eg screenprinting) [4] Usual implementations of electrochemical sen-sors commonly utilize a planar electrode configuration butmicronanoscale patterning of the electrodes provides manytechnical advantages such as a higher surface area and thecontrol over the diffusion profile near the electrode surfaces[5] This surface patterning also allows for more efficient

utilization of functional coatings (eg enzyme or bindingcoatings) on the electrodes [6] Employing these techniquesto decrease the total area needed per sensor along withembedding CMOS electronics underneath the sensor leadsto reducing the system size to practical dimensions [7] Thissize reduction also provides major advantages in reducingindividual device cost and foreign body response to suchdevices when used for in vivo applications [8]

The most common fabrication methods of depositingelectrodes for electrochemical sensors at small (sub-mm) sizescale includes direct Physical Vapor Deposition (PVD) orChemical Vapor Deposition (CVD) These techniques whenused under nonequilibrium conditions can yield patternedsurfaces [9] However the resulting structures are typicallynot suitable for long-term use because of their deformationin liquid environments as a result of large capillary forcesacting on the surface nanostructures in a solution [10]This limitation can be overcome by use of liquid depositiontechniques for example VLS growth [11] porous templates[12] or complex electrochemical plating mechanisms [13]since these methods have better resilience to liquid forcesHowever such deposition approaches are usually not com-patible with the current CMOS fabrication technologies or

Hindawi Publishing CorporationJournal of NanotechnologyVolume 2015 Article ID 467190 13 pageshttpdxdoiorg1011552015467190

2 Journal of Nanotechnology

other vacuum-based fabrication processes Although it hasbeen shown that these alternative methods can be adaptedfor fabrication on wafer scale the overall process is generallycomplex and expensive Often it involves deposition of verythick (10 120583m) metal layers as templates [10] In the casesthat nonvacuum (eg liquid) processing has been appliedto fabricate nanostructures exact control of the geometry atthe nanometer scale has proven difficult High temperaturescorrosive atmospheres or nonuniform deposition due tointernal stresses during deposition often render such bottom-up methods troublesome to scale up with consistency overlarge areas and over many batches for the reproducibleindustrial fabrication of low-impedance electrodes [9]

In this work we present a top-down fabrication method-ology to fabricate nanoscale patterns on electrodes with veryprecise control over the exact geometry and with unifor-mity over large areas Our method uses only vacuum-basedprocessing without the need for making electrical contactsto the devices to be patterned or any exotic liquid-basedprocessing This enables a precise control over the electro-chemical environment surrounding the sensor electrodesNanofabricated silicon pillars are coated with metal to formour electrodes and exhibit excellent mechanical resilienceand successfully resist liquid surface tension forces Wedemonstrate deposition of differentmetals on nanostructuresto render them effective for many different sensing andactuation applications The large and controllable surfacearea of lithographically patterned surfaces provides a simpleand efficient technique to obtain excellent and predictablesensor performance in medical applications such as whenquantitatively determining the concentrations of metabolitesand when detecting DNA [14] We believe that this is the firstdemonstration of metallized nanopillars in fully integratedelectrochemical sensor systems with negligible variation insensor properties over many fabrication batches Metalliza-tion of etched nanostructures can be easily scaled up throughindustrial fabrication approaches to produce high surfacearea sensors in existing micronanofabrication foundriesAnother advantage of metallization of nanostructures is thatthe sensor elements can be shaped in 3D using well-studiedand well-controlled silicon-based fabrication processing Sil-icon etching is a very mature and well-understood fieldand three-dimensional structures can be achieved with greatrepeatability as well as geometric accuracy [15]

2 Device Design

The sensor design to test the effects of surface area enhance-ment on electrochemical performance is based upon athree-electrode electrochemical sensor configuration with anoptional fourth electrode which can be electrically connectedwhen needed The standard three electrodes are the workingelectrode (WE) counter electrode (CE) and reference elec-trode (RE) [16] The optional fourth electrode can be usedas a second working electrode for differential readings orfor background normalization as the sensor ages All of theelectrodes are defined by processing a silicon substrate whichallows vacuum-based nanometer scale silicon processingtechniques to be applied The patterned surface benefits

from the excellent mechanical elasticity and resilience ofnanostructures [17] The surface material of the electrodescan be chosen to match the requirements of a particularapplication Since the sensing mechanism only depends onthe exposed surface layer and since most electrochemicallyactive materials are precious metals [18] depositing a thinmetal layer on top of an underlying silicon substrate iseconomically very beneficial Using only a thin layer alsoprovidesmechanical integrity sincemost of the pillar compo-sition is single crystal silicon The reference electrode for ourelectrochemical measurement consists of AgAgCl thin filmbilayer deposited on the RETheCE is coatedwith Pt formostof our applications and theWEmaterial varies based upon thespecific application but is commonly Pt or Au for our devicesA typical sensor is designed to fit in an area of 500 120583m by500120583m squareThe sensor is connected to large contact padsvia metal contact lines The contact lines are insulated (usinginsulating epoxy eg SU8) to only expose the sensor to testsolutions during sensor testing The contact pads are used toconnect the sensor to a test instrument (eg Potentiostat) toread its electrical output on a computer A typical sensor withcontact pads is depicted in Figure 1

The sensor electrodes need to be coated with spe-cific chemistry (ie ldquofunctionalizedrdquo) to react with specificmolecules for detection in complex biochemical environment[19] To perform glucose sensing in the presence of manysimilar molecules in a complex solution for example thesensor must be coated with a glucose-sensitive layer [20]Many different types of functionalization methods have beendeveloped to convert nanoscale sensors into sensitive andspecific devices [19] For amperometric measurements witha redox enzyme in situ functionalization is a simple andefficient method which allows the application of function-alized materials directly to the sensor surface [21] Thismethod has the benefit that no electrical connections needto be made to the ultra-small scale devices which greatlysimplifies the fabrication However it is important to notethat electrochemically deposited coatings can be controlledmore precisely and can be used for wired sensors [22]

In this work functionalization is achieved either byusing direct immobilization of a hydrogel containing thedesired chemistry (eg glucose-sensitive hydrogel) or bywetting the electrodes in a solution containing the desiredbinding molecules (eg nucleic acid strands) depending onthe application A typical sensor geometry employing basedfunctionalization layer is illustrated in Figure 2

3 Analysis

The main reason for micronanopatterning is to enhancethe sensitivity of the device since it is directly related tothe surface area available for the sensing mechanism Thisadvantage of patterned electrodes over planar electrodes canbe quantified by comparing the surface area of a circular pillarpatterned electrode (119878) as compared to a planar electrode (119878119900)(the derivation for (1) and (2) is given in the Appendix)

119878

119878119900= 1+ 726( 119903

119886)(

ℎ

119886) (1)

Journal of Nanotechnology 3

Sensor electrodesContact pads

Figure 1 Patterned electrochemical sensor design with contact pads

Polymer encapsulation Functionalization matrix

Figure 2 Nanopatterned electrodes coated with a functionalizing (immobilization) matrix

Here 119903 is the radius of the pillars ℎ is the height of thepillars 119886 is the separation between the pillars (center tocenter distance) and the arrangement is hexagonal closedpack form For a standard rectangular array of pillars thissurface area equation becomes

119878

119878119900= 1+ 628( 119903

119886)(

ℎ

119886) (2)

For practical applications the difference between hexagonaland rectangular packing is negligible and therefore we uti-lized rectangular arrangements as these are easier to rescalequickly The theoretical surface area enhancement from suchpatterning is plotted against the size of the pillars in Figure 3Here pillar aspect ratio is fixed (ℎ119903 = 20) to signify the effectof reducing pillar radius on surface area

This shows that higher aspect ratio and higher packingdensity result in higher surface area for a given geometricareaThe exact increase depends upon the scale of patterningand ismostly determined by the application as well as the costof the micro- or nanofabrication The smaller the pillar themore the surface area gain but simultaneously the fabricationbecomes more complex and costly The feature height of thedevices is also limited by the surface tension forces in theliquid which can cause sensor damage depending upon bothmechanical and geometrical properties of the pillars Thedeflection (Δ) of a pillar due to capillary forces is describedby the following equation [10]

Δ =119875ℎ

3

3119864119868 (3)

0 20 40 60 80 1000

10

20

30

40

Pillar radius r (nm)

Surfa

ce ar

ea en

hanc

emen

t (SSo)

Surface area enhancement as function of pillar radius

a = 250nm h = 20 times r

Figure 3 Surface area enhancement due to nanopatterning

Here 119875 is the capillary force ℎ is pillar height 119864 is youngrsquosmodulus and 119868 is second moment of inertia given by

119868 =120587119903

4

4 (4)

Using (3) and (4)

Δ =41198753120587119864119903

(ℎ

119903)

3 (5)


116 120583m

133 120583m122 120583m

428nm

(a) (b)

Figure 4 SEM image of plasma-etched nanopillars demonstrating size-independent etching (a) 1 120583m pillar beside a 50 nm pillar and (b)50 nm pillar array

(a)

151nm

275nm

(b)

Figure 5 Nanopillars after oxidation (a) High voltage imaging to confirm the conformality of the oxide layer (b)

This shows that for a given capillary force surface tension isproportional to third power of pillar aspect ratio (ℎ119903) andis inversely proportional to its Young modulus (119864) Controlof both of these factors is required to have a safe amount ofdeflection due to capillary forces and limit sensor damageControl on youngrsquos modulus is achieved by using high puritymaterials with high Youngrsquos modulus Silicon and Pt groupmetals have Youngrsquos modulus in GPa range in nanostructuresand can be appropriately flexible [17] Aspect ratio control isachieved during fabrication by controlling pattern sizes andetching time as demonstrated in the next section

4 Fabrication

The complete fabrication sequence used to define our pat-terned sensor electrodes consists of the following steps litho-graphic patterning pattern transfer processes (eg etching)interface control methods including deposition of metals andinsulators isolation coatings between electrodes and finallyin some applications functionalization of the metal contactsurface

For nanoscale patterning EUV or electron beam lithog-raphy can provide the small scale feature resolution To be

able to rapidly tune the geometries we have chosen touse electron beam lithography and use PMMA A4 (with amolecular weight of sim950K) to achieve clean liftoff whilestill meeting the required resolution A 50 nm thick aluminamask is sputter coated with a Temescal TES BJD-1800 DCreactive sputter deposition system by depositing aluminumin the presence of oxygen plasma for 5 minutes The roomtemperature silicon plasma etch recipe for nanoscale featuresis described byHenry et al [15]The etch recipewas iterativelyoptimized to achieve uniform etch depth for different pillarwidths and uniform sidewall roughness Etching results areshown in Figure 4 demonstrating uniformity of etch overdifferent pillar sizes and over large arrays

We then thermally oxidized the pillar structures in awafer furnace at 1000∘C for 90minutes followed by 15-minutenitrogen anneal with a gradual return to room temperatureThe results show a very uniform and continuous layer ofoxide as seen in Figure 5 A FEI Sirion 200 scanning electronmicroscope was used for this high contrast imaging The Sicore and the oxide outer layer can be differentiated due to thesecond electron emission imaging contrast between siliconand its oxide

For standard CMOS devices thick top metal (sim46 120583m)aluminum is used for laying out the sensor electrodes


Figure 6 Nanopillars etched in CMOS top metal pads

integrated in the CMOS process itself This aluminum waspatterned using a combination of wet and dry processingtechniques First ma-N 2400 resist was used as either anelectron beam or deep UV resist based upon feature sizeA dilute TMAH based developer (eg MF319) was used forresist development which also results in some etching ofunderlying aluminum This was followed by a Unaxis RIEbased system utilizing a mixture of boron trichloride (BCl

3)

and chlorine (Cl2) plasma for etching rest of aluminumThis

process resulted in very uniform pillar arrays of aluminumas shown in Figure 6

Both silicon and aluminum are not very suitable forelectrochemical sensing directly [23] Hence the top mate-rials were chosen to be more suitable materials dependingupon the particular application For this purpose sputterdeposition of low-impedance metals was used to achieveconformal coatings of Pt group metals High density argonplasma of 20millitorr was used to increase the isotropy of thedeposition A 10 nm Ti adhesion layer was first DC sputterdeposited followed by 100 nm Au or Pt films which wereDC sputtered onto the Ti The deposited film layer grew aconformal coating with uniform thickness on the top sideand base of both pillars and substrate as shown in Figure 7

In our designs the RE consists of a planar AgAgCl elec-trode The AgAgCl bilayer is formed by vacuum depositionof 300 nm of Ag on the RE using liftoff This is followedby low-power RIE chlorine plasma (10W forward power) toconvert a thin (100 nm) top layer into AgClThe compositionof the film is confirmed using SEM and EDX analysis as wellas by a change in color of the electrode surface (from thewhiteldquosilverrdquo color to a brown AgCl color) SEM images of Ag andAgCl films on our electrodes are shown in Figure 8

The completed electrochemical cell needs to be encap-sulated in a material which isolates it from its liquid envi-ronment and leaves only the sensor exposed to the fluidsto be measured This also enables the formation of a reliefwell structure to hold the functionalization chemistry inplace close to the sensor contact We used a thin layer oflithographically patterned SU8 (approximately 2 120583m) as theinsulatorpassivation layer as shown in Figure 9

(a)

(b)

Figure 7 Nanopillars after sputter coating of 50 nm of Au (a) pillararray and (b) pillar array after tweezing to show the metal contactsat the base

Overall this lithographic electrochemical cell fabrica-tion process itself confirms that the nanopatterned elec-trode structure is mechanically robust as it can successfullywithstand high surface tension polymer resist applicationand subsequent processing involving multiple immersionsinto solvents and water Individual liquid drop evaporationtests were also performed on these devices and showed nodestructive effects This demonstrated that we can achievepatterned surfaces with simple vacuum-based processingtechniques

5 Testing and Results

The sensors were tested to quantify the effects of surfacepatterning on electrochemical sensing performance anddetermine if the extra processing incurred for patterning isworth its potential advantages For all cases we used a planarsensor as a reference and compared the patterned sensors tothe reference for their performance

Sensors were tested using a commercial CHI 7051DPotentiostat and an electrochemical test cell with a cell standpositioned on a programmable hot platestirrer A smallmagnetic stirrer was used to allow fast and uniform mixingof test solutions in the background solutionThe backgroundsolution was 001M PBS (pH 74) in all cases A computer-controlled syringe pump (NE-300) was used to introduce


(a) (b)

Figure 8 Thin film reference electrode materials (a) Ag and (b) AgCl

WE CE

RE

SU8

(a)

WE

SU8

(b)

Figure 9 Polymer (SU8) encapsulation around sensors (a) encapsulation around three-electrode sensor and (b) higher resolution viewshowing pillars with SU8 encapsulation surrounding

small volumes of test solutions in a slow flow-cell A depictionof the experimental setup is shown in Figure 10

Results of different electrochemical experiments per-formed on the fabricated devices are summarized in follow-ing subsections

51 Electrochemical Impedance Spectroscopy Electrochemi-cal impedance spectroscopy (EIS) is used to evaluate theimpedance of an electrochemical cell under a given set oftest parameters A small AC signal is applied to the electro-chemical cell on top of a DC bias potential and resulting ACcurrent is measured using the Potentiostat To understandthe expected results from these measurements we used theRandles model of an electrochemical cell which includes theimpedance of the electrodes and the solution modeled as acombination of impedances [16] as illustrated in Figure 11

In this model 119862dl is the double layer capacitance atthe electrode-electrolyte interface 119877ct is the contact (chargetransfer) resistance between the electrode and the electrolyte119885119908is the diffusion limitation (Warburg impedance) from the

bulk to the electrode-electrolyte interface and 119877119904is the series

resistance of the bulk solution between the electrodes

Analytically the electrochemical parameters of the sys-tem depend upon electrode area and the frequency ofoperation For example the double layer capacitance andcontact resistance for an electrode with surface area 119860 aregiven by the following equations [16]

119862dl = 119870 lowast 119860

119877ct =119896119879

119911119902

10038161003816100381610038161003816100381610038161003816

1119894119879

10038161003816100381610038161003816100381610038161003816

(6)

A typical value of 119870 is 10ndash100 120583Fcm2 for most materialsand it is different for different materials 119896 is the Boltzmannconstant 119879 is temperature (Kelvin) 119911 is the ionic charge 119902is the electronic charge and 119894

119879is the electrode current The

solution resistance can be calculated from its resistivity (120588)

and electrode spacing (119871) and overlapping area (119860) [16]

119877119904=120588119871

119860 (7)

For fast voltage changes (eg chronometry or cyclic voltam-metry) current is given by the Cottrell equation as a function


Computer Syringe pump

PotentiostatSoftware

communication

Heaterstirrer Cellstand

Samplefluid

Electrical measurement

Figure 10 Schematic of the electrochemical cell test setup

CdlCdl

Rct

Rs

ZwRct Zw

WE

RE

CE

WW

Figure 11 Randles equivalent electricalmodel of an electrochemicalcell

of time anddiffusion characteristics of the specie(s) of interest[16]

119868 = 119899119865119860119888119900

radic119863

120587119905 (8)

Here 119865 is faraday constant 119863 is the diffusion coefficient 119899is the number of electrons involved in the reaction 119862

119900is the

initial concentration of the specie and 119905 is time If we modelnanopatterned electrodes simply as planar electrodes of thesame total geometric area and ignore the overlap in electricdouble layer (using considerably large greater than 1 nm

spacing between the nanopillars) then we can apply theabove equations that show that the current is proportional tothe electrode surface area and hence the contact resistancedecreases proportionally with increase in electrochemicalsurface area due to the patterning In addition the doublelayer capacitance increases proportionally and the solutionresistance also decreases proportionally to the surface areaTheCE is designed to be an order ofmagnitude larger that theWE so that it does not limit the WE output Hence the cellimpedance would be determined by the WE impedance forpractical applications For simplicity neglecting theWarburgimpedance (diffusion limitations) and assuming that thedouble layer capacitance acts as a simple capacitor the cellimpedance is given by the parameters of the WE [16] as

119885cell =1

1119877ct + 1198952120587119891119862dl+119877119904 (9)

Here 119891 is the frequency of operation in Hertz Since bothresistors are inversely proportional to the surface area andthe capacitance is proportional to the surface area the aboveequation shows that the cell impedance is inversely propor-tional to the surface area of the working electrodes Also thesolution resistance (typically in KΩ range) is generally much

smaller than electrode resistance (typically in MΩ range) forthe electrode sizes used in this work and can be neglected Insuch cases (9) can be further simplified to

119885cell =119877ct

1 + 1198952120587119891119862dl119877ct (10)

Equation (10) is a simple electrical equivalent impedanceof a parallel RC network However in EIS experimentsfrequency (119891) is also a control variable and is not fixed Thisshows that the cell impedance is dependent upon electrodeparameters as well as on the frequency of operation Thefrequency of operation depends upon the actual use case butis mostly near DC for most biosensor applications [24]

In our experiments the frequency of the EIS input ACsignal was swept from 100KHz to 1mHz with a small biasof 10mV using the CHI 7051D Potentiostat Sensors withdifferent working electrodes and varying pillar sizes (10 120583m300 nm and 200 nm) but with the same Pt counter electrodeand AgAgCl reference electrode were tested A simpleplanar electrode was also tested for reference purposes

The result of fitting the EIS data for the different cellsto the Randles model gives us numerical values for thedifferent components of the cell Some experimental results ofimpedance spectroscopy on patterned electrodes are shownin the Nyquist plot format in Figure 12

These results clearly show that as the pillar diameter isreduced (and hence the number of pillars is increased) thereal and imaginary components of the impedance are bothreduced It is also evident that the cell impedance representsan RC circuit as the imaginary component of impedance isnegative The decrease in impedance with frequency can bebetter seen through a Bode plot as shown in Figure 13

This plot shows that the difference between electrodeimpedances is in fact more pronounced at lower frequenciesThis is suitable for most biochemical sensing applications asthe methods of detection in such cases are based upon rel-atively slow voltage changes (eg constant potential amper-ometry cyclic voltammetry) This can also be used for elec-trophysiological applications where smaller electrode sizesare necessary but high electrode impedance is problematic forquality of measurements [25]

From Figure 13 a decrease in impedance from downscal-ing nanostructures is expectable but the measured decreaseis surprisingly more than an order of magnitude in all casesand at 200 nm spacing which reaches more than two ordersof magnitude decrease This is larger than expected from


0 1 2 3 4 50

05

1

15

2

25

times109

times109

Zreal (Ohms)

f

minusZ

imag

inar

y(O

hms)

(a)

Planar 300 nm200 nm

0 1 2 3 4times107

times107

0

1

2

3

4

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(b)

0

1

2

3

4

5

Planar 300 nm200 nm

0 1 2 3 4 5times106

times106

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(c)

Figure 12 Electrochemical impedance spectroscopy results (a)comparison of planar and patterned electrodes (10120583m 300 nmand 200 nm) on the same scale Direction of arrow indicatesincreasing frequency (b) Rescaled view of impedances to highlightmicropatterned electrodes (c) Rescaled view of impedances tohighlight nanopatterned electrodes

a purely geometric surface area increase as predicted by (2)(119878119878119900 = 21) We speculate that this anomaly results from thenondiffusion limited design of the micronanoelectrodes andhigher charge transfer capabilities of such smaller electrodesThere is some similarity between our results and otherrelated works reporting higher charge transfer efficiencies tonanopatterned surfaces [10]

0 2 4 60

2

4

6

8

10

log(f) (Hz)

log(Z

) (O

hms)

Planar 300 nm spacing200 nm spacing

minus2minus4

10120583m

Figure 13 Bode plot of electrode impedance (on loglog scale)

The relation between decreasing impedance and smallernanostructuring will end once the electric double layer ofadjacent nanostructures begins to overlap and then theenhancement in overall effective surface area (electrochem-ical equivalent surface area) will become lower than theactual device surface area This interference happens whenthe nanopillar spacing is comparable to the electric doublelayer thickness which itself is dependent upon the electrolytein the relevant environment However for the commonelectrolytes the electric double layer should only range fromabout 01 nm to 1 nm [26] so there is currently little concernabout this limitation

52 Nucleic Acid Sensing Nucleic acid (eg DNA) sensinghas many biomedical applications including disease diagnos-tics and gene mutation detection [27] Electrochemical DNAsensors are capable of sensitive and selective detection ofDNA strands andmutations through binding (hybridization)reactions using different detection mechanisms [28] Mostmethods of DNA sensing work by attaching ssDNA strand toan electrode as a probeThe probe can have a redox moleculeattached to it and its resulting redox current can provide anindication of the configuration of the probe strand When acomplimentary (target) ssDNA is introduced to the solutionit binds with the probe and changes the configuration of theDNA strand This change moves the redox molecule furtheraway from the surface of the electrode than its resting positionthus causing a change in redox current at the electrode [29]There are different redox species that can be used for thispurpose including methylene blue (MB) and ferrocene (Fc)[30] We chose MB as an indicator for DNA hybridizationas it has been widely reported to have a good conjugationefficiencywithDNAand can create a significant signal changein its different states [29] It has been shown to be effective inmany cases of hybridization based sensors for example fordetection of pathogen DNA [31] and proteins using aptamer[32]

Our probe DNA consists of 17 bases and has a MB redoxmolecule attached at its 51015840 end and a C6 thiol at its 31015840 endThe target DNA is a complimentary strand with 17 basesWe used gold working electrodes (100 nm sputtered goldlayer) for these sensors due to ease of thiol bonding between


the nucleic acid strands and electrode surface The CE wascovered with 100 nm Pt and the RE was an AgAgCl bilayerplanar electrodeTheWEandCEwere patternedwith 250 nmdiameter pillars with 500 nm spacing between pillars Forexperimental protocol we followed the approach previouslydemonstrated by Rowe et al [33] The probe and target DNAwas purchased from Biosearch Technologies Inc in drypowder form The probe DNA was dissolved in PBS solution(pH 74 001M) and its concentration was measured using aNanodrop 2000c spectrophotometer A typical concentrationof 2 120583M of the DNA stock solution was prepared Tris(2-carboxyethyl) phosphine (TCEP) solution was also mixed inPBS to achieve 50mM concentration and was added intothe DNA stock solution (in 2 1 volume ratio between DNAand TCEP solution) to reduce any disulfide bonds in theDNA solution The solution was left at room temperaturefor 20 minutes to complete this reduction The DNA stocksolution was then diluted with PBS to achieve appropriateprobe concentrations (typically 100 nM) and the sensors wereimmersed in this solution for three hours Next the sensorswere immersed in a 2mM mercaptohexanol solution in PBSfor six hours to form a back-filling self-assembled monolayer(SAM) to minimize the formation of direct bonds betweenany target DNA and the gold electrodes Finally these elec-trodes were used in the standard electrochemical test setupconsisting of a beaker on a cell stand and connection to thePotentiostat A background signal was collected by runningsquare wave voltammetry from 0V tominus06V at 100Hz versusthe AgAgCl reference electrode Target DNA solution wasmade by dissolving the DNA powder in PBS measuringthe resulting concentration optically (using the Nanodrop2000c spectrophotometer) and then appropriately diluting itto reach a stock concentration of 2120583M Controlled amounts(100 120583L) of this target DNA solution were then added to thebackground PBS solution (using a syringe pump) to providean overall target concentration of 10 nM in the final solutionThe same voltammetry cycle was repeated on various timeintervals The difference in the peak current before and afterthe addition of the target DNA corresponds to the decreasein redox current of the methylene blue probe as a resultof the change in the morphology of the probe strand dueto hybridization We measured the time dependence of thehybridization process and confirmed excellent sensor sensi-tivity towards the target DNA The results for nanopatternedelectrode are shown in Figure 14

The results indicate that surface nanopatterning increasesthe signal level by decreasing the overall electrochemicalimpedance and by increasing the hybridization efficiencywith larger number of hybridization target sites As a falsepositive test the experiment was repeated with nonspe-cific target DNA confirming that there is no appreciablehybridization or binding in that case

The measured electrical current levels for the patternedsensors are comparable to those measured in a macroscaleplanar electrode (3mm diameter Au electrode) which weused as a reference in this study The following graph(Figure 15) compares the response of a nanopatterned sensorwith the planar sensor It shows that nanopatterned sensorprovides orders of magnitude more responsive compared

02

3

4

5

6

7

8

Potential versus AgAgCl (V)

Curr

ent d

iffer

ence

(A)

No hybridization4-minute hybridization

6-minute hybridization8-minute hybridization

minus01 minus02 minus03 minus04 minus05 minus06

times10minus6

Figure 14 Square wave voltammetric detection of DNA hybridiza-tion

0 5 10

0

1000

2000

3000

Sensing time (min)

Sens

or cu

rren

t diff

eren

ce (n

A)

Planar sensor dataNanopatterned sensor dataLinear fit of nanopatterned data

minus1000

Figure 15 Comparison of nucleic acid hybridization detectionefficiency for planar and nanopatterned sensors

to the planar sensor and that actual enhancement is morethan that just predicted by the increase in surface area asper (2) We speculate that this is similar to the decrease inelectrode impedance as in the EIS experiments describedearlier We suggest that the mechanism of increase in signalis both due to the increase in electrode surface area anda change in the diffusion profile of nucleic acid molecules(3D near a nanopatterned surface versus 2D for a planarsurface) and increase in charge transfer efficiency due to thenanopatterning

These results demonstrate that nanopatterned electrodescan be effectively used for nucleic acid sensing applica-tions where available area is environmentally constrainedSome example applications are system-on-chip devices pointof care diagnostic devices and long-term implants usingaptamers [34]


Figure 16 Glucose sensor after functionalization

53 Glucose Sensing Glucose sensing is an important appli-cation for electrochemical sensors since it can provide clin-ically valuable and accurate results for millions of diabeticpatients [35] We fabricated sensors based upon platinumworking electrodes for glucose measurements The choiceof material was based upon high electrochemical activity ofplatinum formetabolic sensing applications [36]The CEwasalso Pt and the RE was made of AgAgCl The electrodeswere functionalized with glucose oxidase enzyme to achievea glucose selective response as it is the consensus standardenzyme for electrochemical glucose sensors [37] The immo-bilization was performed in situ using BSA based hydrogelas the immobilization matrix The required solutions weremade in 001M PBS with a pH close to that of human bodyfluids (74)The hydrogel used was based upon Bovine SerumAlbumin (BSA) solution and was used to immobilize glucoseoxidase on the electrodes Glutaraldehyde was used as acrosslinking agent for the hydrogel

The experimental procedure for immobilization wasadopted from the literature and iteratively optimized for ourapplication [38] Glucose oxidase (GOx) solution was madeby dissolving 16mg of GOx (Sigma-Aldrich type VII fromAspergillus niger) powder in 10 120583L of PBS The Nanodrop2000c was used to confirm the concentrations of enzymein the resulting solution BSA solution was made in PBSby dissolving 4mg powder in 30 120583l of PBS Glutaraldehyde(25 stock solution) was diluted in PBS to 25 Theenzyme solution was first diluted in PBS by adding 1 partof enzyme solution in 4 parts of PBS The resulting mixturewas mixed with BSA solution PBS in 1 1 ratio by volumeFinally Glutaraldehyde solution was mixed in this mixtureby 3 1 volume ratio between BSA and enzyme solution andGlutaraldehyde Then 1 120583L solution from the final mixturewas pipetted carefully onto the electrochemical sensor Thedevicewas left at room temperature for 10minutes to allow gelformation while the process was continually observed undera microscopeThe sensor was then soaked in PBS for 3 hoursto let any unbound enzyme and BSA dissolve away and resultin stable gel chemistry The sensor after functionalization isshown in Figure 16

The sensor was then dried and placed in the experimentalsetup with the Potentiostat Controlled amounts of glucosestock solution (1M)were added to the background PBS in theelectrochemical cell and the resulting signals were measuredusing the Potentiostat with Cyclic Voltammetry (CV) andamperometry A typical CV scan is performed using voltage

0010203040506

0

1

2


Curr

ent (

A)

0 mM glucose3 mM glucose


minus1

minus2minus01 minus02

times10minus7

Figure 17 In vitro cyclic voltammetric sensing of glucose

from 0 to 06V versus the AgAgCl electrode at a scan rate of001 Vsecond A typical result is shown in Figure 17

The sensor was fabricated with planar electrodes as wellas patterned electrodes (200 nm pillar diameter 500 nmpillar spacing) The response of a nanostructured sensorwith the same electrode geometry as a planar electrode wasmuch higher as shown in Figure 18 Further enhancement ispossible by performing direct immobilization of enzymes onthe electrodes which enhances the electron transfer efficiencyfrom the enzyme to the electrode [39]

The glucose sensor response shows saturation effectsdue to mismatch between glucose and oxygen levels in thesolution [40]The response can be optimized by addingmorelayers of suitable membranes to the sensor chemistry stack[40] and is the focus of our future efforts To test specificitydifferent solutions of PBS and pH buffers (with pH sim7) wereadded as controls but did not cause any appreciable changein the sensor response Overall the sensitivity increase dueto nanopatterning was more than two orders of magnitudeas compared to the planar electrode This response is similarto what we observed in the earlier described EIS and nucleicacid sensing and likely follows from the same geometric anddiffusion reasons

These results indicate that the miniaturized sensors canprovide good sensitivity towards glucose in the physiologicalrange (2ndash20mM) Such sensors when combined with appro-priate circuitry wireless powering and telemetry techniquescan form the basis of ultra-small (mm scale) size and cost-effective integrated sensing platforms [41] The resultingsmall size can pave the way for such systems to be usedfor long-term in vivo applications [42] thus revolutionizingthe field of continuous glucose monitoring and other similarsensing applications

6 Summary and Conclusions

In this paper we demonstrated the advantages of integratedelectrochemical sensors based upon micronanopatternedelectrodes over traditional planar sensors We showed thatwhen the die area is limited such patterning techniques


Planar sensor outputSpline fitting

0 5 10 15 20Glucose concentration (mM)

0

10

20

30

40

50

Sens

or cu

rren

t (nA

)

(a)

0 5 10 15 200

1000

2000

3000

4000

5000

6000

Glucose concentration (mM)

Sens

or cu

rren

t (nA

)

Planar sensor dataNanopatterned sensor dataShape-preserving interpolant fitting

(b)

Figure 18 Response curve of glucose sensor (a) Planar sensor (b)Patterned sensor

can overcome the sensitivity limitations of electrochemi-cal sensors Hence adequate signal to noise ratio can beachieved in different applications resulting in miniaturiza-tion of overall sensing platforms We also demonstrate thatsuch patterning can be achieved using standard fabricationtechniques thus limiting the need of special methods whichcan add to cost and reliability of the fabrication processThis has the advantage that such electrodes can be createdusing common fabrication facilities with currently runninginfrastructure which allows for low device fabrication costsand highmanufacturing reproducibility Such patterning aidsin the goal of shrinking electrode sizes due to measurementenvironment constraints and allows for smaller total platformfootprints We demonstrated that these methods can be usedwith standard substrates (eg Si) as well as metal electrodeson CMOS substrates but other material systems may also beapplicable

We validated the effect of surface patterning in decreasingelectrode impedance for a given electrode size We showedthat the smaller the scale of patterning is the denser theresulting patterns are and hence the more pronouncedis the enhancement in electrode properties (decrease inimpedance) We also provided examples of DNA and glucosesensing to demonstrate the sensitivity advantage of suchpatterned electrodes but broader applications are possiblesuch as in electrophysiology Furthermore we argue thatusing such techniques on standard electronic platforms (likeCMOS) is very favorable for scaling production of a sensingplatform using integrated CMOS-MEMS facilities which arebecoming more and more cost effective with time [43] Thiswould minimize the failure modes related with productionof large number of sensors using prior methods which aremore difficult to scale and need more specialized facilities forproper manufacturing control

Our future work is focused on broadening the range ofapplications for such electrodes as well as in optimizing thesensor chemistry to result in more efficient sensors for real-world applications

Appendix

(a) For patterned electrode with hexagonal arrangement ofpillars with height ℎ radius 119903 and separation 119886 surface areaenhancement is due to the area of pillars which is a product oftheir circumference and height Hence total surface area forthe patterned electrode is sum of the rectangular geometricarea and the circumferential surface area The surface areafor such hexagonal packed structure can be calculated usinga unit cell as described here [10]

The surface area of planar electrode is given by

119878119900 =3radic32

1198862 (A1)

The surface area of patterned electrode is given by

119878 =3radic32

1198862+ 6120587119903ℎ (A2)

Hence the area ratio would be given by

119878

119878119900= 1+ 4120587119903ℎ

radic311198862

= 1+ 726( 119903119886)(

ℎ

119886) (A3)

(b) For rectangular electrode surface area of planarelectrode is given by (assuming length 119871 of both sides)

119878119900 = 1198712 (A4)

For patterned electrode with rectangular arrangement ofpillars with height ℎ radius 119903 and separation 119886 surface areaenhancement is due to the area of pillars which is a productof their circumference and height The circumferential areadepends upon number of pillars which is given by ratio ofeach side to the pillar separation (center to center distance)

119878 = 1198712

+ 2120587119903ℎ1198712

1198862 (A5)



119878

119878119900= 1+ 2120587119903ℎ 1

1198862= 1+ 628( 119903

119886)(

ℎ

119886) (A6)

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The fabrication procedures in this work were performed atthe Kavli Nanoscience Institute at the California Institute ofTechnology This work was supported by Sanofi A G underGrant no D094502653189 The authors would like to thankMr Mehmet Sencan for his help with functionalization andtesting

References

[1] C M Li H Dong X Cao J Luong and X Zhang ImplantableElectrochemical Sensors for Biomedical andClinical ApplicationsProgress Problems and Future Possibilities vol 14 BenthamScience Publishers 2007

[2] S Ayers K D Gillis M Lindau and B A Minch ldquoDesign of aCMOS potentiostat circuit for electrochemical detector arraysrdquoIEEE Transactions on Circuits and Systems I Regular Papers vol54 no 4 pp 736ndash744 2007

[3] X-J Huang and Y-K Choi ldquoChemical sensors based onnanostructured materialsrdquo Sensors and Actuators B Chemicalvol 122 no 2 pp 659ndash671 2007

[4] E Jubete O A Loaiza E Ochoteco J A Pomposo H Grandeand J Rodrıguez ldquoNanotechnology a tool for improved perfor-mance on electrochemical screen-printed (bio)sensorsrdquo Journalof Sensors vol 2009 Article ID 842575 13 pages 2009

[5] A K Wanekaya W Chen N V Myung and A MulchandanildquoNanowire-based electrochemical biosensorsrdquo Electroanalysisvol 18 no 6 pp 533ndash550 2006

[6] J H T Luong R S Brown and W H Scouten ldquoEnzyme orprotein immobilization techniques for applications in biosensordesignrdquo Trends in Biotechnology vol 13 no 5 pp 178ndash185 1995

[7] MMujeeb-U-Rahman and A S M H Nazari ldquoAn implantablecontinuous glucose monitoring microsystem in 018 120583mCMOSrdquo in Proceedings of the VLSI Syposium HonoluluHawaii USA June 2014

[8] W K Ward E P Slobodzian K L Tiekotter and M DWood ldquoThe effect of microgeometry implant thickness andpolyurethane chemistry on the foreign body response to sub-cutaneous implantsrdquo Biomaterials vol 23 no 21 pp 4185ndash41922002

[9] M Lee Y Jeon T Moon and S Kim ldquoTop-down fabricationof fully CMOS-compatible silicon nanowire arrays and theirintegration into CMOS inverters on plasticrdquo ACS Nano vol 5no 4 pp 2629ndash2636 2011

[10] G Zhang ldquoDesign and fabrication of 3D skyscraper nanos-tructures and their application as electrodesrdquo in BiosensorsNew Perspectives in Biosensors Technology and Applications PAndrea Serra Ed InTech 2011

[11] S M U Ali T Aijazi K Axelsson O Nur and M WillanderldquoWireless remote monitoring of glucose using a functionalized

ZnO nanowire arrays based sensorrdquo Sensors vol 11 no 9 pp8485ndash8496 2011

[12] A Huczko ldquoTemplate-based synthesis of nanomaterialsrdquoApplied Physics A Materials Science and Processing vol 70 no4 pp 365ndash376 2000

[13] Y-J Lee D-J Park J-Y Park and Y Kim ldquoFabrication andoptimization of a nanoporous platinum electrode and a non-enzymatic glucose micro-sensor on siliconrdquo Sensors vol 8 no10 pp 6154ndash6164 2008

[14] R C Barry Y Lin J Wang G Liu and C A TimchalkldquoNanotechnology-based electrochemical sensors for biomon-itoring chemical exposuresrdquo Journal of Exposure Science andEnvironmental Epidemiology vol 19 no 1 pp 1ndash18 2009

[15] M D Henry S Walavalkar A Homyk and A SchererldquoAlumina etchmasks for fabrication of high-aspect-ratio siliconmicropillars and nanopillarsrdquo Nanotechnology vol 20 no 25Article ID 255305 2009

[16] A J Bard and L R Faulkner Electrochemical Methods Funda-mentals and Applications JohnWiley amp Sons 2nd edition 2001

[17] A Heidelberg L T Ngo BWu et al ldquoA generalized descriptionof the elastic properties of nanowiresrdquo Nano Letters vol 6 no6 pp 1101ndash1106 2006

[18] D Grieshaber R MacKenzie J Voros and E Reimhult ldquoElec-trochemical biosensorsmdashsensor principles and architecturesrdquoSensors vol 8 no 3 pp 1400ndash1458 2008

[19] R A Sheldon ldquoEnzyme immobilization the quest for optimumperformancerdquo Advanced Synthesis and Catalysis vol 349 no 8-9 pp 1289ndash1307 2007

[20] S J Updike and G P Hicks ldquoThe enzyme electroderdquo Naturevol 214 no 5092 pp 986ndash988 1967

[21] S Zimmermann D Fienbork A W Flounders and D Liep-mann ldquoIn-device enzyme immobilization wafer-level fabrica-tion of an integrated glucose sensorrdquo Sensors and Actuators BChemical vol 99 no 1 pp 163ndash173 2004

[22] P N Bartlett and D J Caruana ldquoElectrochemical immobi-lization of enzymes Part V Microelectrodes for the detec-tion of glucose based on glucose oxidase immobilized in apoly(phenol) filmrdquo Analyst vol 117 no 8 pp 1287ndash1292 1992

[23] S Park H Boo and T D Chung ldquoElectrochemical non-enzymatic glucose sensorsrdquo Analytica Chimica Acta vol 556no 1 pp 46ndash57 2006

[24] S-M Park and J-S Yoo ldquoElectrochemical impedance spec-troscopy for better electrochemical measurementsrdquo AnalyticalChemistry vol 75 no 21 pp 455Andash461A 2003

[25] V S Polikov P A Tresco and W M Reichert ldquoResponse ofbrain tissue to chronically implanted neural electrodesrdquo Journalof Neuroscience Methods vol 148 no 1 pp 1ndash18 2005

[26] S G Real J R Vilche andA J Arvia ldquoThe impedance responseof electrochemically roughened platinum electrodes Surfacemodeling and roughness decayrdquo Journal of ElectroanalyticalChemistry vol 341 no 1-2 pp 181ndash195 1992

[27] T G DrummondMGHill and J K Barton ldquoElectrochemicalDNA sensorsrdquo Nature Biotechnology vol 21 no 10 pp 1192ndash1199 2003

[28] J J Gooding ldquoElectrochemical DNA hybridization biosensorsrdquoElectroanalysis vol 14 no 17 pp 1149ndash1156 2002

[29] S O Kelley J K Barton N M Jackson and M G HillldquoElectrochemistry ofmethylene blue bound to aDNA-modifiedelectroderdquo Bioconjugate Chemistry vol 8 no 1 pp 31ndash37 1997

[30] D Kang X Zuo R Yang F Xia K W Plaxco and R J WhiteldquoComparing the properties of electrochemical-based DNA


sensors employing different redox tagsrdquo Analytical Chemistryvol 81 no 21 pp 9109ndash9113 2009

[31] A Erdem K Kerman B Meric U S Akarca and M OzsozldquoNovel hybridization indicator methylene blue for the elec-trochemical detection of short DNA sequences related to thehepatitis B virusrdquo Analytica Chimica Acta vol 422 no 2 pp139ndash149 2000

[32] G S Bang S Cho and B-G Kim ldquoA novel electrochemicaldetectionmethod for aptamer biosensorsrdquoBiosensorsampBioelec-tronics vol 21 no 6 pp 863ndash870 2005

[33] A A Rowe R J White A J Bonham and K W Plaxco ldquoFab-rication of electrochemical-DNA biosensors for the reagentlessdetection of nucleic acids proteins and small moleculesrdquoJournal of Visualized Experiments no 52 article 2922 2011

[34] J Liu Z Cao and Y Lu ldquoFunctional nucleic acid sensorsrdquoChemical Reviews vol 109 no 5 pp 1948ndash1998 2009

[35] S Vaddiraju D J Burgess I Tomazos F C Jain and FPapadimitrakopoulos ldquoTechnologies for continuous glucosemonitoring current problems and future promisesrdquo Journal ofDiabetes Science and Technology vol 4 no 6 pp 1540ndash15622010

[36] G-H Wu X-H Song Y-F Wu X-M Chen F Luo andX Chen ldquoNon-enzymatic electrochemical glucose sensorbased on platinum nanoflowers supported on graphene oxiderdquoTalanta vol 105 pp 379ndash385 2013

[37] S Ferri K Kojima and K Sode ldquoReview of glucose oxidasesand glucose dehydrogenases a birdrsquos eye view of glucose sensingenzymesrdquo Journal of Diabetes Science and Technology vol 5 no5 pp 1068ndash1076 2011

[38] N F de Rooji M Koudelka-Hep and D J Strike ldquoActivationof rayonpolyester cloth for protein immobilizationrdquo in Immo-bilization of Enzymes and Cells G F Bickerstaff Ed vol 1 pp77ndash82 Humana Press Totowa NJ USA 1997

[39] M V Pishko A C Michael and A Heller ldquoAmperometricglucose microelectrodes prepared through immobilization ofglucose oxidase in redox hydrogelsrdquo Analytical Chemistry vol63 no 20 pp 2268ndash2272 1991

[40] S Vaddiraju A Legassey YWang et al ldquoDesign and fabricationof a high-performance electrochemical glucose sensorrdquo Journalof Diabetes Science and Technology vol 5 no 5 pp 1044ndash10512011

[41] M Mujeeb-U-Rahman D Adalian M Sencan and A SchererldquoNanofabrication techniques for fully integrated sensing plat-formsrdquo in Nanotech 2013 pp 73ndash76 NSTI 2013

[42] J C Pickup F Hussain N D Evans and N Sachedina ldquoInvivo glucose monitoring the clinical reality and the promiserdquoBiosensors andBioelectronics vol 20 no 10 pp 1897ndash1902 2005

[43] AWitvrouw ldquoCMOS-MEMS integration why how andwhatrdquoin Proceedings of the Annual Conference on CAD (ComputerAided Design) (ICCAD rsquo06) San Jose Calif USA November2006

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


other vacuum-based fabrication processes Although it hasbeen shown that these alternative methods can be adaptedfor fabrication on wafer scale the overall process is generallycomplex and expensive Often it involves deposition of verythick (10 120583m) metal layers as templates [10] In the casesthat nonvacuum (eg liquid) processing has been appliedto fabricate nanostructures exact control of the geometry atthe nanometer scale has proven difficult High temperaturescorrosive atmospheres or nonuniform deposition due tointernal stresses during deposition often render such bottom-up methods troublesome to scale up with consistency overlarge areas and over many batches for the reproducibleindustrial fabrication of low-impedance electrodes [9]

In this work we present a top-down fabrication method-ology to fabricate nanoscale patterns on electrodes with veryprecise control over the exact geometry and with unifor-mity over large areas Our method uses only vacuum-basedprocessing without the need for making electrical contactsto the devices to be patterned or any exotic liquid-basedprocessing This enables a precise control over the electro-chemical environment surrounding the sensor electrodesNanofabricated silicon pillars are coated with metal to formour electrodes and exhibit excellent mechanical resilienceand successfully resist liquid surface tension forces Wedemonstrate deposition of differentmetals on nanostructuresto render them effective for many different sensing andactuation applications The large and controllable surfacearea of lithographically patterned surfaces provides a simpleand efficient technique to obtain excellent and predictablesensor performance in medical applications such as whenquantitatively determining the concentrations of metabolitesand when detecting DNA [14] We believe that this is the firstdemonstration of metallized nanopillars in fully integratedelectrochemical sensor systems with negligible variation insensor properties over many fabrication batches Metalliza-tion of etched nanostructures can be easily scaled up throughindustrial fabrication approaches to produce high surfacearea sensors in existing micronanofabrication foundriesAnother advantage of metallization of nanostructures is thatthe sensor elements can be shaped in 3D using well-studiedand well-controlled silicon-based fabrication processing Sil-icon etching is a very mature and well-understood fieldand three-dimensional structures can be achieved with greatrepeatability as well as geometric accuracy [15]

2 Device Design

The sensor design to test the effects of surface area enhance-ment on electrochemical performance is based upon athree-electrode electrochemical sensor configuration with anoptional fourth electrode which can be electrically connectedwhen needed The standard three electrodes are the workingelectrode (WE) counter electrode (CE) and reference elec-trode (RE) [16] The optional fourth electrode can be usedas a second working electrode for differential readings orfor background normalization as the sensor ages All of theelectrodes are defined by processing a silicon substrate whichallows vacuum-based nanometer scale silicon processingtechniques to be applied The patterned surface benefits

from the excellent mechanical elasticity and resilience ofnanostructures [17] The surface material of the electrodescan be chosen to match the requirements of a particularapplication Since the sensing mechanism only depends onthe exposed surface layer and since most electrochemicallyactive materials are precious metals [18] depositing a thinmetal layer on top of an underlying silicon substrate iseconomically very beneficial Using only a thin layer alsoprovidesmechanical integrity sincemost of the pillar compo-sition is single crystal silicon The reference electrode for ourelectrochemical measurement consists of AgAgCl thin filmbilayer deposited on the RETheCE is coatedwith Pt formostof our applications and theWEmaterial varies based upon thespecific application but is commonly Pt or Au for our devicesA typical sensor is designed to fit in an area of 500 120583m by500120583m squareThe sensor is connected to large contact padsvia metal contact lines The contact lines are insulated (usinginsulating epoxy eg SU8) to only expose the sensor to testsolutions during sensor testing The contact pads are used toconnect the sensor to a test instrument (eg Potentiostat) toread its electrical output on a computer A typical sensor withcontact pads is depicted in Figure 1

The sensor electrodes need to be coated with spe-cific chemistry (ie ldquofunctionalizedrdquo) to react with specificmolecules for detection in complex biochemical environment[19] To perform glucose sensing in the presence of manysimilar molecules in a complex solution for example thesensor must be coated with a glucose-sensitive layer [20]Many different types of functionalization methods have beendeveloped to convert nanoscale sensors into sensitive andspecific devices [19] For amperometric measurements witha redox enzyme in situ functionalization is a simple andefficient method which allows the application of function-alized materials directly to the sensor surface [21] Thismethod has the benefit that no electrical connections needto be made to the ultra-small scale devices which greatlysimplifies the fabrication However it is important to notethat electrochemically deposited coatings can be controlledmore precisely and can be used for wired sensors [22]

In this work functionalization is achieved either byusing direct immobilization of a hydrogel containing thedesired chemistry (eg glucose-sensitive hydrogel) or bywetting the electrodes in a solution containing the desiredbinding molecules (eg nucleic acid strands) depending onthe application A typical sensor geometry employing basedfunctionalization layer is illustrated in Figure 2

3 Analysis

The main reason for micronanopatterning is to enhancethe sensitivity of the device since it is directly related tothe surface area available for the sensing mechanism Thisadvantage of patterned electrodes over planar electrodes canbe quantified by comparing the surface area of a circular pillarpatterned electrode (119878) as compared to a planar electrode (119878119900)(the derivation for (1) and (2) is given in the Appendix)

119878

119878119900= 1+ 726( 119903

119886)(

ℎ

119886) (1)







119878

119878119900= 1+ 628( 119903

119886)(

ℎ

119886) (2)



Δ =119875ℎ

3

3119864119868 (3)

0 20 40 60 80 1000

10

20

30

40


Surfa

ce ar

ea en

hanc

emen

t (SSo)





119868 =120587119903

4

4 (4)

Using (3) and (4)

Δ =41198753120587119864119903

(ℎ

119903)

3 (5)


116 120583m

133 120583m122 120583m

428nm

(a) (b)


(a)

151nm

275nm

(b)



4 Fabrication









3)






(a)

(b)







(a) (b)


WE CE

RE

SU8

(a)

WE

SU8

(b)









119862dl = 119870 lowast 119860

119877ct =119896119879

119911119902

10038161003816100381610038161003816100381610038161003816

1119894119879

10038161003816100381610038161003816100381610038161003816

(6)





119877119904=120588119871

119860 (7)





communication


Samplefluid



CdlCdl

Rct

Rs

ZwRct Zw

WE

RE

CE

WW



119868 = 119899119865119860119888119900

radic119863

120587119905 (8)


119900is the



119885cell =1

1119877ct + 1198952120587119891119862dl+119877119904 (9)



119885cell =119877ct

1 + 1198952120587119891119862dl119877ct (10)








0 1 2 3 4 50

05

1

15

2

25

times109

times109

Zreal (Ohms)

f

minusZ

imag

inar

y(O

hms)

(a)

Planar 300 nm200 nm

0 1 2 3 4times107

times107

0

1

2

3

4

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(b)

0

1

2

3

4

5

Planar 300 nm200 nm

0 1 2 3 4 5times106

times106

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(c)



0 2 4 60

2

4

6

8

10

log(f) (Hz)

log(Z

) (O

hms)


minus2minus4

10120583m









02

3

4

5

6

7

8


Curr

ent d

iffer

ence

(A)




times10minus6


0 5 10

0

1000

2000

3000

Sensing time (min)

Sens

or cu

rren

t diff

eren

ce (n

A)


minus1000









0010203040506

0

1

2


Curr

ent (

A)



minus1


times10minus7











0

10

20

30

40

50

Sens

or cu

rren

t (nA

)

(a)

0 5 10 15 200

1000

2000

3000

4000

5000

6000


Sens

or cu

rren

t (nA

)


(b)





Appendix



119878119900 =3radic32

1198862 (A1)


119878 =3radic32

1198862+ 6120587119903ℎ (A2)


119878

119878119900= 1+ 4120587119903ℎ

radic311198862

= 1+ 726( 119903119886)(

ℎ

119886) (A3)


119878119900 = 1198712 (A4)


119878 = 1198712

+ 2120587119903ℎ1198712

1198862 (A5)



119878

119878119900= 1+ 2120587119903ℎ 1

1198862= 1+ 628( 119903

119886)(

ℎ

119886) (A6)



Acknowledgments


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









119878

119878119900= 1+ 628( 119903

119886)(

ℎ

119886) (2)



Δ =119875ℎ

3

3119864119868 (3)

0 20 40 60 80 1000

10

20

30

40


Surfa

ce ar

ea en

hanc

emen

t (SSo)





119868 =120587119903

4

4 (4)

Using (3) and (4)

Δ =41198753120587119864119903

(ℎ

119903)

3 (5)


116 120583m

133 120583m122 120583m

428nm

(a) (b)


(a)

151nm

275nm

(b)



4 Fabrication









3)






(a)

(b)







(a) (b)


WE CE

RE

SU8

(a)

WE

SU8

(b)









119862dl = 119870 lowast 119860

119877ct =119896119879

119911119902

10038161003816100381610038161003816100381610038161003816

1119894119879

10038161003816100381610038161003816100381610038161003816

(6)





119877119904=120588119871

119860 (7)





communication


Samplefluid



CdlCdl

Rct

Rs

ZwRct Zw

WE

RE

CE

WW



119868 = 119899119865119860119888119900

radic119863

120587119905 (8)


119900is the



119885cell =1

1119877ct + 1198952120587119891119862dl+119877119904 (9)



119885cell =119877ct

1 + 1198952120587119891119862dl119877ct (10)








0 1 2 3 4 50

05

1

15

2

25

times109

times109

Zreal (Ohms)

f

minusZ

imag

inar

y(O

hms)

(a)

Planar 300 nm200 nm

0 1 2 3 4times107

times107

0

1

2

3

4

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(b)

0

1

2

3

4

5

Planar 300 nm200 nm

0 1 2 3 4 5times106

times106

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(c)



0 2 4 60

2

4

6

8

10

log(f) (Hz)

log(Z

) (O

hms)


minus2minus4

10120583m









02

3

4

5

6

7

8


Curr

ent d

iffer

ence

(A)




times10minus6


0 5 10

0

1000

2000

3000

Sensing time (min)

Sens

or cu

rren

t diff

eren

ce (n

A)


minus1000









0010203040506

0

1

2


Curr

ent (

A)



minus1


times10minus7











0

10

20

30

40

50

Sens

or cu

rren

t (nA

)

(a)

0 5 10 15 200

1000

2000

3000

4000

5000

6000


Sens

or cu

rren

t (nA

)


(b)





Appendix



119878119900 =3radic32

1198862 (A1)


119878 =3radic32

1198862+ 6120587119903ℎ (A2)


119878

119878119900= 1+ 4120587119903ℎ

radic311198862

= 1+ 726( 119903119886)(

ℎ

119886) (A3)


119878119900 = 1198712 (A4)


119878 = 1198712

+ 2120587119903ℎ1198712

1198862 (A5)



119878

119878119900= 1+ 2120587119903ℎ 1

1198862= 1+ 628( 119903

119886)(

ℎ

119886) (A6)



Acknowledgments


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




116 120583m

133 120583m122 120583m

428nm

(a) (b)


(a)

151nm

275nm

(b)



4 Fabrication









3)






(a)

(b)







(a) (b)


WE CE

RE

SU8

(a)

WE

SU8

(b)









119862dl = 119870 lowast 119860

119877ct =119896119879

119911119902

10038161003816100381610038161003816100381610038161003816

1119894119879

10038161003816100381610038161003816100381610038161003816

(6)





119877119904=120588119871

119860 (7)





communication


Samplefluid



CdlCdl

Rct

Rs

ZwRct Zw

WE

RE

CE

WW



119868 = 119899119865119860119888119900

radic119863

120587119905 (8)


119900is the



119885cell =1

1119877ct + 1198952120587119891119862dl+119877119904 (9)



119885cell =119877ct

1 + 1198952120587119891119862dl119877ct (10)








0 1 2 3 4 50

05

1

15

2

25

times109

times109

Zreal (Ohms)

f

minusZ

imag

inar

y(O

hms)

(a)

Planar 300 nm200 nm

0 1 2 3 4times107

times107

0

1

2

3

4

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(b)

0

1

2

3

4

5

Planar 300 nm200 nm

0 1 2 3 4 5times106

times106

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(c)



0 2 4 60

2

4

6

8

10

log(f) (Hz)

log(Z

) (O

hms)


minus2minus4

10120583m









02

3

4

5

6

7

8


Curr

ent d

iffer

ence

(A)




times10minus6


0 5 10

0

1000

2000

3000

Sensing time (min)

Sens

or cu

rren

t diff

eren

ce (n

A)


minus1000









0010203040506

0

1

2


Curr

ent (

A)



minus1


times10minus7











0

10

20

30

40

50

Sens

or cu

rren

t (nA

)

(a)

0 5 10 15 200

1000

2000

3000

4000

5000

6000


Sens

or cu

rren

t (nA

)


(b)





Appendix



119878119900 =3radic32

1198862 (A1)


119878 =3radic32

1198862+ 6120587119903ℎ (A2)


119878

119878119900= 1+ 4120587119903ℎ

radic311198862

= 1+ 726( 119903119886)(

ℎ

119886) (A3)


119878119900 = 1198712 (A4)


119878 = 1198712

+ 2120587119903ℎ1198712

1198862 (A5)



119878

119878119900= 1+ 2120587119903ℎ 1

1198862= 1+ 628( 119903

119886)(

ℎ

119886) (A6)



Acknowledgments


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






3)






(a)

(b)







(a) (b)


WE CE

RE

SU8

(a)

WE

SU8

(b)









119862dl = 119870 lowast 119860

119877ct =119896119879

119911119902

10038161003816100381610038161003816100381610038161003816

1119894119879

10038161003816100381610038161003816100381610038161003816

(6)





119877119904=120588119871

119860 (7)





communication


Samplefluid



CdlCdl

Rct

Rs

ZwRct Zw

WE

RE

CE

WW



119868 = 119899119865119860119888119900

radic119863

120587119905 (8)


119900is the



119885cell =1

1119877ct + 1198952120587119891119862dl+119877119904 (9)



119885cell =119877ct

1 + 1198952120587119891119862dl119877ct (10)








0 1 2 3 4 50

05

1

15

2

25

times109

times109

Zreal (Ohms)

f

minusZ

imag

inar

y(O

hms)

(a)

Planar 300 nm200 nm

0 1 2 3 4times107

times107

0

1

2

3

4

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(b)

0

1

2

3

4

5

Planar 300 nm200 nm

0 1 2 3 4 5times106

times106

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(c)



0 2 4 60

2

4

6

8

10

log(f) (Hz)

log(Z

) (O

hms)


minus2minus4

10120583m









02

3

4

5

6

7

8


Curr

ent d

iffer

ence

(A)




times10minus6


0 5 10

0

1000

2000

3000

Sensing time (min)

Sens

or cu

rren

t diff

eren

ce (n

A)


minus1000









0010203040506

0

1

2


Curr

ent (

A)



minus1


times10minus7











0

10

20

30

40

50

Sens

or cu

rren

t (nA

)

(a)

0 5 10 15 200

1000

2000

3000

4000

5000

6000


Sens

or cu

rren

t (nA

)


(b)





Appendix



119878119900 =3radic32

1198862 (A1)


119878 =3radic32

1198862+ 6120587119903ℎ (A2)


119878

119878119900= 1+ 4120587119903ℎ

radic311198862

= 1+ 726( 119903119886)(

ℎ

119886) (A3)


119878119900 = 1198712 (A4)


119878 = 1198712

+ 2120587119903ℎ1198712

1198862 (A5)



119878

119878119900= 1+ 2120587119903ℎ 1

1198862= 1+ 628( 119903

119886)(

ℎ

119886) (A6)



Acknowledgments


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b)


WE CE

RE

SU8

(a)

WE

SU8

(b)









119862dl = 119870 lowast 119860

119877ct =119896119879

119911119902

10038161003816100381610038161003816100381610038161003816

1119894119879

10038161003816100381610038161003816100381610038161003816

(6)





119877119904=120588119871

119860 (7)





communication


Samplefluid



CdlCdl

Rct

Rs

ZwRct Zw

WE

RE

CE

WW



119868 = 119899119865119860119888119900

radic119863

120587119905 (8)


119900is the



119885cell =1

1119877ct + 1198952120587119891119862dl+119877119904 (9)



119885cell =119877ct

1 + 1198952120587119891119862dl119877ct (10)








0 1 2 3 4 50

05

1

15

2

25

times109

times109

Zreal (Ohms)

f

minusZ

imag

inar

y(O

hms)

(a)

Planar 300 nm200 nm

0 1 2 3 4times107

times107

0

1

2

3

4

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(b)

0

1

2

3

4

5

Planar 300 nm200 nm

0 1 2 3 4 5times106

times106

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(c)



0 2 4 60

2

4

6

8

10

log(f) (Hz)

log(Z

) (O

hms)


minus2minus4

10120583m









02

3

4

5

6

7

8


Curr

ent d

iffer

ence

(A)




times10minus6


0 5 10

0

1000

2000

3000

Sensing time (min)

Sens

or cu

rren

t diff

eren

ce (n

A)


minus1000









0010203040506

0

1

2


Curr

ent (

A)



minus1


times10minus7











0

10

20

30

40

50

Sens

or cu

rren

t (nA

)

(a)

0 5 10 15 200

1000

2000

3000

4000

5000

6000


Sens

or cu

rren

t (nA

)


(b)





Appendix



119878119900 =3radic32

1198862 (A1)


119878 =3radic32

1198862+ 6120587119903ℎ (A2)


119878

119878119900= 1+ 4120587119903ℎ

radic311198862

= 1+ 726( 119903119886)(

ℎ

119886) (A3)


119878119900 = 1198712 (A4)


119878 = 1198712

+ 2120587119903ℎ1198712

1198862 (A5)



119878

119878119900= 1+ 2120587119903ℎ 1

1198862= 1+ 628( 119903

119886)(

ℎ

119886) (A6)



Acknowledgments


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






communication


Samplefluid



CdlCdl

Rct

Rs

ZwRct Zw

WE

RE

CE

WW



119868 = 119899119865119860119888119900

radic119863

120587119905 (8)


119900is the



119885cell =1

1119877ct + 1198952120587119891119862dl+119877119904 (9)



119885cell =119877ct

1 + 1198952120587119891119862dl119877ct (10)








0 1 2 3 4 50

05

1

15

2

25

times109

times109

Zreal (Ohms)

f

minusZ

imag

inar

y(O

hms)

(a)

Planar 300 nm200 nm

0 1 2 3 4times107

times107

0

1

2

3

4

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(b)

0

1

2

3

4

5

Planar 300 nm200 nm

0 1 2 3 4 5times106

times106

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(c)



0 2 4 60

2

4

6

8

10

log(f) (Hz)

log(Z

) (O

hms)


minus2minus4

10120583m









02

3

4

5

6

7

8


Curr

ent d

iffer

ence

(A)




times10minus6


0 5 10

0

1000

2000

3000

Sensing time (min)

Sens

or cu

rren

t diff

eren

ce (n

A)


minus1000









0010203040506

0

1

2


Curr

ent (

A)



minus1


times10minus7











0

10

20

30

40

50

Sens

or cu

rren

t (nA

)

(a)

0 5 10 15 200

1000

2000

3000

4000

5000

6000


Sens

or cu

rren

t (nA

)


(b)





Appendix



119878119900 =3radic32

1198862 (A1)


119878 =3radic32

1198862+ 6120587119903ℎ (A2)


119878

119878119900= 1+ 4120587119903ℎ

radic311198862

= 1+ 726( 119903119886)(

ℎ

119886) (A3)


119878119900 = 1198712 (A4)


119878 = 1198712

+ 2120587119903ℎ1198712

1198862 (A5)



119878

119878119900= 1+ 2120587119903ℎ 1

1198862= 1+ 628( 119903

119886)(

ℎ

119886) (A6)



Acknowledgments


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0 1 2 3 4 50

05

1

15

2

25

times109

times109

Zreal (Ohms)

f

minusZ

imag

inar

y(O

hms)

(a)

Planar 300 nm200 nm

0 1 2 3 4times107

times107

0

1

2

3

4

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(b)

0

1

2

3

4

5

Planar 300 nm200 nm

0 1 2 3 4 5times106

times106

Zreal (Ohms)

minusZ

imag

inar

y(O

hms)

10120583m

(c)



0 2 4 60

2

4

6

8

10

log(f) (Hz)

log(Z

) (O

hms)


minus2minus4

10120583m









02

3

4

5

6

7

8


Curr

ent d

iffer

ence

(A)




times10minus6


0 5 10

0

1000

2000

3000

Sensing time (min)

Sens

or cu

rren

t diff

eren

ce (n

A)


minus1000









0010203040506

0

1

2


Curr

ent (

A)



minus1


times10minus7











0

10

20

30

40

50

Sens

or cu

rren

t (nA

)

(a)

0 5 10 15 200

1000

2000

3000

4000

5000

6000


Sens

or cu

rren

t (nA

)


(b)





Appendix



119878119900 =3radic32

1198862 (A1)


119878 =3radic32

1198862+ 6120587119903ℎ (A2)


119878

119878119900= 1+ 4120587119903ℎ

radic311198862

= 1+ 726( 119903119886)(

ℎ

119886) (A3)


119878119900 = 1198712 (A4)


119878 = 1198712

+ 2120587119903ℎ1198712

1198862 (A5)



119878

119878119900= 1+ 2120587119903ℎ 1

1198862= 1+ 628( 119903

119886)(

ℎ

119886) (A6)



Acknowledgments


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







02

3

4

5

6

7

8


Curr

ent d

iffer

ence

(A)




times10minus6


0 5 10

0

1000

2000

3000

Sensing time (min)

Sens

or cu

rren

t diff

eren

ce (n

A)


minus1000









0010203040506

0

1

2


Curr

ent (

A)



minus1


times10minus7











0

10

20

30

40

50

Sens

or cu

rren

t (nA

)

(a)

0 5 10 15 200

1000

2000

3000

4000

5000

6000


Sens

or cu

rren

t (nA

)


(b)





Appendix



119878119900 =3radic32

1198862 (A1)


119878 =3radic32

1198862+ 6120587119903ℎ (A2)


119878

119878119900= 1+ 4120587119903ℎ

radic311198862

= 1+ 726( 119903119886)(

ℎ

119886) (A3)


119878119900 = 1198712 (A4)


119878 = 1198712

+ 2120587119903ℎ1198712

1198862 (A5)



119878

119878119900= 1+ 2120587119903ℎ 1

1198862= 1+ 628( 119903

119886)(

ℎ

119886) (A6)



Acknowledgments


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials








0010203040506

0

1

2


Curr

ent (

A)



minus1


times10minus7











0

10

20

30

40

50

Sens

or cu

rren

t (nA

)

(a)

0 5 10 15 200

1000

2000

3000

4000

5000

6000


Sens

or cu

rren

t (nA

)


(b)





Appendix



119878119900 =3radic32

1198862 (A1)


119878 =3radic32

1198862+ 6120587119903ℎ (A2)


119878

119878119900= 1+ 4120587119903ℎ

radic311198862

= 1+ 726( 119903119886)(

ℎ

119886) (A3)


119878119900 = 1198712 (A4)


119878 = 1198712

+ 2120587119903ℎ1198712

1198862 (A5)



119878

119878119900= 1+ 2120587119903ℎ 1

1198862= 1+ 628( 119903

119886)(

ℎ

119886) (A6)



Acknowledgments


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






0

10

20

30

40

50

Sens

or cu

rren

t (nA

)

(a)

0 5 10 15 200

1000

2000

3000

4000

5000

6000


Sens

or cu

rren

t (nA

)


(b)





Appendix



119878119900 =3radic32

1198862 (A1)


119878 =3radic32

1198862+ 6120587119903ℎ (A2)


119878

119878119900= 1+ 4120587119903ℎ

radic311198862

= 1+ 726( 119903119886)(

ℎ

119886) (A3)


119878119900 = 1198712 (A4)


119878 = 1198712

+ 2120587119903ℎ1198712

1198862 (A5)



119878

119878119900= 1+ 2120587119903ℎ 1

1198862= 1+ 628( 119903

119886)(

ℎ

119886) (A6)



Acknowledgments


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





119878

119878119900= 1+ 2120587119903ℎ 1

1198862= 1+ 628( 119903

119886)(

ℎ

119886) (A6)



Acknowledgments


References






















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials

























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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