Asymmetric NanochannelâIonchannel Hybrid for Ultrasensitive and

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Asymmetric Nanochannel-Ionchannel Hybrid for Ultrasensitive and Label-free Detection of Copper Ions in Blood Xiao-Ping Zhao, Shan-Shan Wang, Muhammad Rizwan Younis, Chen Wang, and Xing-Hua Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03818 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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Analytical Chemistry

Asymmetric Nanochannel-Ionchannel Hybrid for Ultrasensitive and Label-free Detection of Copper Ions in Blood Xiao-Ping Zhao,1,2 Shan-Shan Wang,1 Muhammad Rizwan Younis,2 Chen Wang,*1 Xing-Hua Xia*2

1

Key Laboratory of Biomedical Functional Materials, School of Science, State Key

Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China 2

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry

and Chemical Engineering, Nanjing University, Nanjing 210093, China *To whom correspondence should be addressed. E-mail: [email protected]; [email protected]

ABSTRACT：Nanochannel/nanopre based analysis methods attracts increasingly interests in recent years due to the exquisite ability of revealing the changes in molecular volume. In this work, a highly asymmetric nanochannel-ionchannel hybrid coupled with electrochemical technique was developed for copper ions (Cu2+) detection. The polyglutamic acid (PGA) is modified in the nanochannels array of porous anodic alumina (PAA). When different concentrations of Cu2+ are introduced into the nanochannel-ionchannel hybrid in neutral environment, Cu2+-PGA chelation reaction occurs, resulting in varied current-potential (I-V) properties of the nanochannel-ionchannel hybrid. When PAA is immersed in a low pH solution, the PGA-Cu2+ complex dissociates. Based on the changed ionic current, the label-free assay of Cu2+ and regenerated application of the constructed platform can be achieved. Due to unique mass transfer property of the nanochannel-ionchannel hybrid, combing with the highly amplified ionic current magnitude of nanochannels array, significantly increased assay sensitivity was achieved as expected. To evaluate the applicability of the present methodology for detecting Cu2+ in a real sample, the Cu2+ content in real blood samples were analyzed. Results demonstrated that the present method shows

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excellent selectivity with high sensitivity towards Cu2+ detection in real blood samples. Keywords: copper ion, porous anodic alumina, nanochannel-ionchannel hybrid, electrochemical technique, blood

INTRODUCTION As an essential trace element for organisms, copper ion (Cu2+) is one of the most important cofactors for constituting metalloproteins, which modulates protein functions as well as produces numerous enzymes critical for life.1 However, overloading or deficiency of Cu2+ will give rise to a disturbance of the cellular homeostasis, resulting in serious damage to the central nervous system and disorders associated with neurodegenerative diseases (e.g., Wilson’s diseases and Alzheimer’s disease).2 In recent years, elevated levels of Cu2+ have also been found to cause many types of human cancers as well as infant liver damage.3 The National Research Council suggests the daily allowance of copper ranges from 1.5 to 3.0 mg for adults, 1.5 to 2.5 mg for children, and 0.4 to 0.6 mg for infants.4 Thus, there is an urgent need to develop convenient and specific ion sensors for the sensitive detection of Cu2+ in the environmental matrix and biological fluids. Up to now, various sensor systems have been developed for Cu2+ detection at trace quantity levels in various sample, such as atomic absorption spectroscopy,5 inductively coupled plasma mass spectroscopy,6 high-performance liquid chromatography,7 colorimetric analysis,8 fluorescence9,10 and nanostructures based techniques.11,12 These methods greatly push forward the progress of Cu2+ detection in different samples. However, these conventional methods usually require considerable quantities of samples, which would become a large challenge when the target amount is rare and limited. In recently years, nanopore/channel has been ongoing interest in construction of biosensors and chemical analytical devices.13-21 Among the particular attributes, nanopore/channel possesses exquisite ability of revealing the changes in molecular volume by measurable ionic current, which is of particularly importance for label-free moleculars/ions detection. For example, Jiang’s group have designed a series of single


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nanopore-based

methods

for

chemo/biosensors

and

bioassays

including

biomolecules,15 metal ions,16 glucose21 and chiral molecules.22 These methods open up

new

avenues

in

developing

highly-sensitive

sensors

based

on

nanopore/nanochannel. Unfortunately, the ability to analyze the trace amounts of sample at low concentration still remains a challenge. To achieve a lower detection limit and amplify the ionic current response, expensive electronic detection equipment such as patch-clamp and other commercial devices are usually required. Compared to single nanopre/channel, nanochannels array of porous anodic alumina (PAA) membrane possess the perfect chemical or mechanical stability, high pore densities, and flexibility in terms of shape and size,23-30 which can not only amplify the ionic current by several orders of magnitude, but also reduce the background noise, and enable excellent detection sensitivity. Using PAA coupled with electrochemical detection technique, analysis including protein,28,30 DNA,25,31 metal ions,26 cancer biomarker,32,33 and Amyloid β aggregation kinetics24 have been successfully detected. This new nanochannels array provides a novel and simple platform for label-free and ultrasensitive detection of molecules/ions and also monitoring molecular recognition process. However, previous reports using PAA mainly focused on the porous layer. Recently, the existence of ion channels in the barrier layer of PAA with size ranging from 0 to 0.8 nm was confirmed.34 Using this special hybrid structure and unique mass transfer property, highly efficiently protein trapping and circulating tumor cells capturing can be achieved.34,35 Actually, this nanochannel-ionchannel hybrid structure has strong geometry-asymmetry, which will result in an unique ionic rectification phenomenon.36 Exploring the mass transfer properties and potential applications of this nanochannel-ionchannel hybrid is expected to bring new chances in sensing, energy conversion and purification technologies. Herein, we for the first time make use of this nanochannel-ionchannel hybrid structure for label-free and ultrasensitive detection of Cu2+ in blood. Polyglutamic acid (PGA, the chemical structure is shown Scheme S1) is used as the probe to be modified in the nanochannels array. As shown in Scheme 1a, in the presence of Cu2+, the oxygen atom of the free carboxyl group and the nitrogen atom of the amide group



of P PGA can binnd with the Cu2+ to forrm Cu2+-PG GA chelation n in aqueouss solution at a pH 7-8..37 The Cu2++-PGA chelation have rrelative larg ger volume,, leading too a different free masss transfer region with hin the nannochannels for ions traansfer. Theerefore a vaaried ioniic current in the preesence of Cu2+ can be expectted. Using a home-m made elecctrochemicaal cell (Scheeme 1b), thee I-V properrty of the naanochannel--ionchannell can be m monitored in real-timee (Scheme 1c). Furtheermore, thee as-prepareed method was founnd to be reggenerable for fo Cu2+ dettections by simple imm mersion of PPAA in low w pH soluution. Resullts show th hat differentt concentraations of Cu u2+ leads too different ionic i currrent responnse and also o ionic recctification behavior. b Using U the ppresent metthod, ultraalow conceentration off Cu2+ as llow as 0.1ffM can be facilely annd successffully deteected.

Sch heme 1. (a) Illustration n of the Cuu2+ detection n principle. (b) Schema matic diagram m of the I-V measurrement setup. (c) The II-V propertiies of PAA under diffeerent condittions withh a scan ratte of 100 mV/s. m Blue line: additiion of Cu2++; Red line: PGA modified arraay nanochannnels; Black k line: pure PAA memb brane.


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EXPERIMENTAL SECTION Materials and reagents. Polyglutamic acid (PGA, Mw>10000) was purchased from Sigma Aldrich. Potassium hydroxide, hydrogen peroxide (30% H2O2), zinc surfate heptahydizte (ZnSO4), magnesium sulfate heptahydrate (MgSO4) and copper sulfate pentahydrate (CuSO4) were purchased from Nanjing Chemical Reagent Co., Ltd. Barium chloride dehydrate (BaCl2) and calcium chloride anhydrous (CaCl2) were purchased from Xilong Science Co., Ltd. Iron (П) sulfate heptahydrate (FeSO4), phosphocromic acid(H3PO4), and Tin(П) chloride were from Sinopharm Chemical Reagent Co., Ltd. Oxalic acid dehydrate, acetone, chromic acid (H2CrO4) and potassium chloride (KCl) were purchased from Shanghai Ling Feng Chemical Reagent Co., Ltd. Silica gel films were purchased from Shanghai Zhang's silicone rubber products Co., Ltd. All reagents were of analytical grade and were used as received. Deionized water with a resistivity of 18.2 MΩ/cm was used in all the above experiments. Instrumentation. The morphology of the prepared nanochannel-ionchannel hybrid structure and the cells capture were characterized using a scanning electron microscope (SEM, S-4800, Japan). UV-vis absorbance was recorded on a U-3000 spectrophotometer. Fourier-transform infrared (FTIR) spectroscopy was performed on a Nicolet 6700 model 912A0637. The electrochemical detection was performed in 10 mM KCl solution on an electrochemical workstation (CHI660E, Chenhua, China) with two Ag/AgCl electrodes as the anode and cathode. Fabrication of Cu2+ responsive nanochannel-ionchannel hybrid. The whole process for fabrication of Cu2+ responsive nanochannel-ionchannel hybrid is schematically shown in Scheme 2. First, PAA were synthesized using self-organizing electrochemical anodization processes.38 Briefly, a high purity aluminum (Al) foil was used as a starting material. Before anodization, Al substrates were degreased in acetone with ultrasound for 10 min. Subsequently, the samples were rinsed with water and etched in 1.0 M KOH and finally the sample was rinsed ultrasonically with distilled water. The PAA layers were fabricated by a two-step anodizing carried out in



0.3 M oxalic acid a at 25°C C under a constant po otential of 40V. 4 The dduration of first anodization waas 30 min in n oxalic acidd. The as-prrepared sam mples were tthen submeerged intoo a mixture of 5 wt % H3PO4 and 1.5 wt % H2CrO4 at 60 °C for 400 min to rem move the oxide layerrs. Subsequ uently, the dduration off second ano odization w was 4 h and d the otheer factors were w carried out under tthe same ex xperimental conditions as were useed in the first step. After ano odization, thhe chemicaal etching in Tin (П)) chloride was perfformed in order o to remove alum minum subsstrate. In th he control eexperiment,, the barrrier layer iss detached using 5% H 3PO4 aqueeous solutio on. Finally, the clean PAA P mem mbrane wass hydroxylaated in boilled hydrogeen peroxide (30% H2O 2) at 98-10 00℃ for 0.5 h to gennerated abu undant –OH H groups. Fo or physical absorption a oof PGA in PAA P nanochannel walls, w the porous p layerr of PAA membranes m s was immeersed into 15.8 μmool/L PGA soolution for 2 h. In the presence off Cu2+, the Cu2+-PGA cchelation fo orms withhin the nanoochannels.

Sch heme 2. Schhematic illusstration of th the fabricatiion process for Cu2+ ressponsive PA AA.

Elettrochemicaal measurem ment. The measuremeent of I–V curve c was pperformed using u CHII 660E eleectrochemiccal workstaation. For this purpo ose, the as--prepared PAA P mem mbrane wass clamped between b twoo silica gel films and then t placedd between tw wo 2 mL homemadee half cells for electrocchemical deetection. 160 00 mL aqueeous electro olyte on of (10 mM KCl) was filled in both hallves of the cell. Then, different cconcentratio Cu22+ solution were w added d into the tw wo half cellls, incubatiing for 8000s at 25℃. The


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Ag/AgCl electrodes were inserted into each half-cell solution to obtain a transmembrane potential and the ionic current flowing through the pore was measured. In order to record the I–V curves, a linear sweep voltammetry signal from − 0.8 V to + 0.8 V was used. Analysis of blood samples. Blood samples from a healthy volunteer (24 years old) were drawn from the vein into tubes containing sodium citrate. The blood collecting procedures were performed in compliance with relevant laws and institutional guidelines. The blood sample preparation generally followed the procedure reported earlier.39 Briefly, 1 mL blood was diluted with 8 mL of DI water and then treated with 1 mL of concentrated HNO3 for 2 h. The sample was briefly centrifuged at 8000 rpm for 12 min. The supernatant was diluted with DI water, adjust the pH value to pH=7 by KOH and analyzed using the present approach. Aliquots of blood samples (1 mL) were spiked with 0, 10-6, 10-2, 106 μM of standard Cu2+ solutions (1 mL). The spiked samples (50 μL) were then added to 1600 μL 10 mM KCl solution to detect. Analysis of tap water samples. The tap water samples collected in our lab were filtered through membrane with 0.22 μm. After being spiked with different concentration of Cu2+ (the same processing method as in the blood samples), the samples were tested according to the above protocol.

RESULTS AND DISCUSSION Characterization of Cu2+ responsive PAA. The morphology of the fabricated nanochannel-ionchannel hybrid was characterized by SEM. It can be observed that the nanochannel diameter of large base side is about 40 nm (Figure 1a). The hexagon of the barrier layer (small tip side) is about 100 nm (Figure 1b) with numerous ionchannels in it. Regular cylinder nanochannels array parallel to each other is clearly shown (Figure 1c). The thickness of the whole membrane is estimated to be 50 μm (Figure 1d). The I-V properties were used to characterize the modification of PGA. As reference, the I-V property of pure PAA was detected shown as the black curve in Figure 1e. Due to the asymmetric structure of the nanochannel-ionchannel hybrid, the channel possesses higher resistance when the ions are driven from the base to tip than



from the tip to the base, resulting in an obvious ionic rectification (ICR) behavior.36 Thus, the ICR was adopted to investigate the ionic transport behavior after modification of PGA. As shown in Figure S1, after modification of PGA, the ICR ratio (I-0.8/I+0.8, where I-0.8 is the magnitude of ionic current recorded at - 0.8 V, I+0.8 is the magnitude of ionic current recorded at + 0.8 V) increases due to the increased charge density of modified PGA (negatively charged). However, in the present of Cu2+, PGA chelates with the positive charge of Cu2+, leading to the decreased negative charge density and ICR ratio. In addition, after modification of PGA, due to the reduction of effective channels diameter in the nanochannel, the ionic current of hybrid lowered, which confirmed that PGA was successfully absorbed onto the nanochannels surface. To further verify the presence of PGA in PAA membrane, UV-vis spectra was performed. According to the reference, the visible absorption maximum of the Cu2+-PGA complex was found in the visible range around 700-800 nm when the complex was prepared by mixing Cu2+ and PGA solutions.37 As shown Figure 1f, the PAA membrane (red line) and PGA (pink line) did not shown the absorption peak at 725 nm, while a clear peak at 725 nm appeared (blue line) in the present of Cu2+. The results were in accordance with the reported reference. FTIR was also used to probe the modification of PGA by monitoring the absorption of amide І and П vibration using pure membrane as the reference. As shown in Figure 1g, the amide І band (1673 cm-1) was attributed to the C=O stretching vibration of the peptide linkage in the peptide background. The amide П band (1627 cm-1) was due to the N-H bending and C-N stretching. The above results in Figure 2 demonstrate the successful modification of PGA in nanochannels.


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Figu ure 1. SEM M images off PAA (a: toop, b: bottom m, c: cross section, d: tthe whole cross c secttion). (e) Thhe I-V propeerties of PA AA in 10 mM M KCl undeer different conditions with a sccan rate of 100 mV/s. Blue line: addition off Cu2+; Red line: PGA modified array a nanochannels; Black line: pure PAA m membrane. (f) UV-vis absorption spectra of pure PAA A membranee (red line),, PGA modiified PAA (p pink line) and a Cu2+ preesent PAA (blue ( linee). (g) FTIR spectrum of o PGA moddified PAA with w pure PAA PA as the rreference.

Dettection of C Cu2+. To sh how the pootential of th he proposed method ffor detectio on of Cu22+ concentraation, a grad dient seriess of concenttrations of Cu2+ was uused as sam mples for quantificattion. The Cu C 2+-PGA rrecognition kinetics was w first invvestigated. The resuult is shownn in Figure S2. This deemonstratess that the Cu u2+-PGA chhelation reaaches equilibrium within w 800 s. Then th the electrocchemical liinear sweep ep voltamm metry techhnique was used to reecord the I--V profiles after equilibrium in tthe presencce of diffe ferent Cu2+ concentratio c ons, and thee results aree shown in Figure F 2a. SSurprisingly y, the supeerlow conceentration off Cu2+ as low w as 3.37 x 10-10 μM caan be succeessfully deteected usinng the preseent platform m. To clearrly show th he changes of I-V curvves in diffeerent



concentration ranges, Figure 2a is separately shown in Figure 2b-d representing for low, medium and high concentrations of Cu2+ respectively. It is clearly found that upon addition of Cu2+, the ionic currents decreases dramatically with the increase of Cu2+ concentration. The decreased ionic current suggests an increasing steric hindrance for ions transport in nanochannels. It has been previously proved that the oxygen atom of the free carboxyl group and the nitrogen atom of the amide group of PGA can bind with the Cu2+ to form Cu2+-PGA chelation in pH 7-8 environment.37 Therefore, the presence of Cu2+ results in formation of Cu2+-PGA chelation, which will lead to the decreased free transport space in nanochannels, and accordingly the dropped ionic current through the nanochannel-ionchannel hybrid. With the Cu2+ concentration increases, a much less ionic current is expected. The control experiment of a PGA-free PAA, the presence of 10 μM Cu2+ shows nearly no change in the ionic current compared to the bare PAA (Figure S3), indicating the formation of Cu2+-PGA chelation is the key factor for Cu2+ detection in the present method. In addition, symmetric nanochannels array (PAA membrane without ionchannel) was used to detect Cu2+ concentration using the same PGA-modified method. The result is shown in Figure S4. It can be seen that for the same Cu2+ concentration range, the ionic currents nearly overlap each other, suggesting a relative lower detection sensitivity. It was therefore manifested that the detection limit of Cu2+ can be greatly improved by utilizing the present asymmetric nanochannel-ionchannel hybrid. To compare the performance of the device, Cu2+ ions were added in only one side of PAA membrane, and the detection was performed as described above. It was found that no obvious change in the device performance occurs for different Cu2+ ions addition modes (Figure S5). However, a much longer time is needed for PGA- Cu2+ recognition equilibrium when Cu2+ ions were added in only one side (Figure S6). That means a longer time will be required for analytes detection if only one side is used. Therefore in the present work, the Cu2+ ions were added on both sides of the PAA membrane, which enables a faster detection rate.


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Figu ure 2. (a) The T I-V pro ofiles of thee PGA mod dified nanocchannel-ionnchannel hy ybrid for different concentrations of Cu2++ from low concentrattion to highh concentraation (Insset: the enlaarged part of the overlaapping curves from -0.8 8 V to -4.0 V). (b) Thee I-V proffiles for thee low Cu2++ concentrattion range. (c) The I-V V profiles ffor the med dium Cu22+ concentration range. (d) The I-V V profiles forr the high Cu C 2+ concenttration rang ge.

To reveal the t relationship betweeen the ionicc current an nd the Cu2++ concentration, oncentration ns is display ayed in Figu ure 3 the ionic currennt values ass a functionn of Cu2+ co wn in Figurre 3a, the cu urrent valuee decreases with and Figure S7. As a wholee trend show Cu22+ increases. Figure 3b b-d show thhe relationsships of thrree differennt concentraation rangges (low, medium, m and d high) resppectively. A very intereesting phenoomenon occcurs. For every sepaarated conceentration raange, the cu urrent values show lineear to logarithm of Cu2+ conccentration. The detecttion limit was down n to 0.1fM M. The results monstrates that t the pro oposed plattform using g nanochann nel-ionchannnel hybrid and dem elecctrochemicaal techniquee provides aan ultrasenssitive and label-free m method for Cu C 2+



deteection.

2 Figu ure 3. (a) The T currentt change veersus logariithm of Cu2+ concentraations. low (b),

meddiun (c) annd high (d)) Cu2+ conncentrationss (C as thee concentraation of Cu2+). Caliibration currve of the present p deviice for low (b, inset), medium m (c, inset) and high (d, iinset) Cu2+determinatio d on.

a Selectiivity of Cu u2+ detectio on. The rev versibility oof the proposed Revversibility and metthod towardd Cu2+ detecction is evalluated. Figu ure 4a show ws the variattion of the ionic i currrent values over differeent cycles oof reversiblee binding an nd unbindinng of Cu2+ with the PGA. As described d ab bove, the ooxygen atom m of the freee carboxyll group and d the nitroogen atom of the amiide group oof PGA can n bind with h Cu2+, form ming Cu2+-P PGA chellation in neuutral enviro onment (pH 7). When PAA P is imm mersed in a llow pH solu ution (pH H 2), the protons can displace d thee chelated Cu C 2+ of PG GA-Cu2+ coomplex, and d the PGA A-Cu2+ coomplex disssociates.37 This rev versible ch hange provvides potential regeenerated appplication off the construucted platform. Selectivity is anotheer importannt factor in developiing sensorss for pracctical


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application. Too investigaate the sellectivity off the PGA-modified PAA for Cu C 2+ 2 deteection, seveeral represen ntative metaal ions inclu uding Ba2+, Ca2+, Mg22+, Fe2+, Zn2+ as

the interferents were inv vestigated uusing the same s metho od. As shoown Figure 4b, 2 addition of Ba2+ , Ca2+, Mg g2+, Fe2+, Znn2+ does no ot result in obvious o chan ange in the ionic i

currrent values (I0: the currrent value of the pure PAA, I: the t current value of PGAmoddified PAA in the preseence of Cu22+ or other interfering mental m ions)), indicating g the exceellent selecctivity of the t propos ed method d toward Cu C 2+ detectition. The good g seleectivity of Cu C 2+ was due to the stroong affinity of cupric io ons bindingg to PGA probes. 2 Com mpared to other metaal ions inccluding Fe2+ , Zn2+, Ca C 2+, Ba2+ and Mg2+, the

disssociation coonstant of Cu u2+ binding to PGA waas much low wer.19

Figu ure 4. (a) The ionic current vallues at -0.8 8 V at pH= =2 and pH= =7 versus Cu C 2+ deteection cyclee numbers. Cu2+ conceentration: 24 40 μM. (b) Selectivityy of Cu2+ (0 0.337 fM)) detection over o other representativve interferin ng metal ion ns (10mM).

Dettection of C Cu2+ in bloo od. To the eend, the app plicability of o the presennt methodology for detecting C Cu2+ in a reeal sample w was furtherr evaluated. After diffeerent amoun nt of Cu22+ were addeed into the blood sampples, the ion nic current dropped obbviously (Figure 5a).. The ionic current at -0.8 V verssus different samples are a displayeed in Figuree 5b. Bassed on the standard s currve in Figurre 3, the meeasured Cu2+ amount w were calculaated, as iillustrated inn Table 1.IIt is shown that the measured m vaalues for diifferent sam mples withh known am mounts of Cu2+ show wed good reecoveries of o 86%-1044%. The results dem monstrated that t the pressent methodd had strong g anti-interfference abiliity toward Cu C 2+



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deteection in reaal blood sam mples. To show the t generaliity of the prresent method platform m towards C Cu2+ detectio on in reall samples, thhe Cu2+ con ntent in tap water was also detecteed using thee same metthod. Thee results werre shown in n Figure S8 and Table S1, confirm ming the exccellent accu uracy and high sensittivity of the present appproach for Cu C 2+ concen ntration deteermination.

Figu ure 5. (a) The T I-V pro ofiles of diffferent conccentrations of Cu2+ in blood samp ples. (Sam mple 1: noo additionall Cu2+ in bblood; Samp ple 2: addeed 5 x 10-77 μM in bllood; Sam mple 3: addeed 5 x 10-3 μM μ in bloodd; Sample 4: 4 added 5 x 105 μM in blood). (b) The ioniic current att -0.8 V verssus differennt blood sam mples.

Taable 1 Resuults of the deetection of C Cu2+ in real blood samp ples. Saample

Added Cu C 2+ (μM)

μM) Measured Cu2+ (μ

( Recovery (%)

Saample 1

0

1.14 x 10-7

/

Saample 2

5 x 10-7

5.44 x 10-7

86.1

Saample 3

5 x 10 1 -3

4.82 x 10-3

96.4

Saample 4

5 x 10 1 5

5.19 5 x 105

103.8

CO ONCLUSIO ON d off Cu2+ has been b Inn summary, a strategy for ultrasennsitive and label-free detection propposed usingg nanochan nnel-ionchannnel hybrid d integrated d with an eelectrochem mical deteector. Due to t the uniqu ue mass trannsfer properrty togetherr with the hhighly amplified currrent magnituude of array y nanochannnels array, trace t level of o Cu2+ as loow as 0.337 7 fM


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can be successfully detected. The detection selectivity and reversibility were also investigated. Results show that the present method shows a good selectivity with ultrasensitivity and good reversibility towards Cu2+ detection. The practicality of our present method was validated by the analyses of blood samples and tap water. This simple and cost-effective system appears to hold great practical potential for the detection of heavy metal ions in biological samples.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the grants from the National Natural Science Foundation of China (21327902, 21575163, 21635004) and the Natural Science Foundation of Jiangsu Province (BK20151437). Supporting Information Available: Supporting Information is available free of charge from the Analytical Chemistry home page (http://pubs.acs.org/journal/ancham).

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