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Sensors and Actuators B 261 (2018) 441–450

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

jo ur nal home page: www.elsev ier .com/ locate /snb

sandwiched electroanalysis method for probing Anthrax DNAsased on glucose-induced gold growth and catalytic coupling ofyramine using gold-mineralized glucose oxidase

ua Wang ∗,1, Liyan Zhang, Yao Jiang, Lijun Chen, Zhiqiang Duan, Xiaoxia Lv, Shuyun Zhunstitute of Medicine and Materials Applied Technologies, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu City, Shandongrovince 273165, PR China

r t i c l e i n f o

rticle history:eceived 5 August 2017eceived in revised form1 December 2017ccepted 21 January 2018

eywords:old nanoparticles

a b s t r a c t

Gold nanoparticles was doped into the protein matrix of glucose oxidase (GOx) by the in-site biomineral-ization route, yielding the GOx with mineralized gold (GOx-Gold) showing the double catalysis activitiesof GOx and peroxidase-like gold catalysis. A magnetic separation-based detection method was thus tai-lored for the sandwiched electroanalysis of Anthrax DNAs using GOx-Gold and tyramine linker as theprobe labels. After the DNA hybridization reactions, the tyramine-mediated linking of DNA capture anddetection probes was conducted through gold-catalytic oxidization of tyramines labelled at the probeterminuses, followed by the glucose-triggered gold growth catalyzed by GOx. Highly amplified electro-

lucose oxidaseouble catalysis activitiesyramine-labelledlucose-triggered gold growthNA detection

chemical output of gold signals was thus achieved toward the ultrasensitive detection of DNAs in blood,with the detection limit down to ∼0.10 fM. Also, the discrimination of DNAs with single-base mutationcould be expected. Importantly, such a biomimic gold mineralization route can be tailored for remold-ing various enzymes with improved intrinsic catalysis and electrocatalysis, thus promising the wideapplications in the catalysis, biosensing, and biomedical fields.

© 2018 Elsevier B.V. All rights reserved.

. Introduction

Recent decades have witnessed the increasing applications ofarious enzymes as the labels for the catalytic signal amplifica-ion for the highly sensitive detections. It is widely recognized thathe catalysis-active centers of enzymes are commonly inaccessi-le, since they are either located close to the electrically insulatedrotein shells of enzymes like horseradish peroxidase (HRP) [1,2],r embedded deep in their glycoprotein sheath like glucose oxi-ase (GOx) [3–6]. Establishment of an efficient electron transferetween enzymes and their supports like the electrodes is vitalor the development of various enzymatic catalysis-based biosen-ors (or bioelectronics) [7–9], catalysts [10,11], and biofuel cells12,13]. Aiming to build up the electrical contacts toward the enzy-

atic redox centers, historically, the use of electron mediators

14,15], the modification of enzymes with redox relays [16,17],nd the incorporation of enzymes in redox-active polymer matri-es [18,19] were commonly practiced, but received the limited

∗ Corresponding author.E-mail address: huawangqfnu@126.com (H. Wang).

1 http://wang.qfnu.edu.cn.

ttps://doi.org/10.1016/j.snb.2018.01.171925-4005/© 2018 Elsevier B.V. All rights reserved.

success in improving the electron transportation of enzymes. Inrecent years, a variety of efforts have been devoted to the develop-ment of direct electron transfer (DET) between the redox enzymesand electrode surfaces by using enzymatic labels of nano-sizedmaterials, typically as gold nanoparticles (NPs) [20,21] and carbonnanotubes (CNTs) [6,22–27]. Nevertheless, the so obtained DETs ofmost enzymes are still compromised, since it is hard for the labelledor coated nanomaterials to electrically plug into the catalysis-activesites of enzymes that are embedded deeply aforementioned. Betterenzymatic DETs have been alternatively proposed by the reconsti-tution of apo-enzymes with cofactors that are functionalized withsome conductive nanomaterials (i.e., gold NPs and CNTs) or redoxtethers [6,26,28,29]. For example, Willner et al. [6] extracted thecatalysis-active center from GOx to be functionalized with goldNPs and further reconstituted with the apo-enzyme yielding theenzyme mimics with the improved electrocatalysis. Takashi and co-workers reconstituted myoglobin with the prosthetic groups thatwere bound with cationic cytochrome c to produce the highly cat-alytic complex [30]. Our group also developed a kind of peroxidase

mimics with the enhanced catalysis and electrocatalysis by recon-stituting hemoglobin with the gold-remolded active centers [31].While these pioneering methods can greatly improve the DETs ofsome enzymes, their practical applicability may be substantially

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42 H. Wang et al. / Sensors and

imited by the complicated experimental procedure. Furthermore,he biomineralization route has emerged as a facile and highlyfficient way for the synthesis of various inorganic materials likeold NPs by using some biological materials such as proteins andnzymes [21,32–37]. For example, Zhang’s group employed HRPo synthesize gold nanoclusters (NCs) with strong fluorescencehrough the biomineralization process for H2O2 sensing [38]. GoldCs were also in site incorporated into alkaline phosphatase by

he protein-mediated biomineralization for a catalytic amplifica-ion technique for the gene analysis [21]. In addition, some noble

etal nanomaterials with small sizes can intrinsically present theeroxidase-like catalysis activities [39–41], thus promising thextensive catalysis applications.

Moreover, many modern biosensors have been established touantify DNAs to date [42,43], especially those by way of theandwiched DNA hybridization and enzyme catalysis-based signalmplification. However, the detection sensitivities of these sand-iched analysis methods can still be trapped generally by the

imited signal amplification, due to that the hybridized doublehains, which can be formed either between targets and the cap-ure probes or between targets and the detection probes, may note robust enough to withstand the enzyme-catalytic signal amplifi-ation at a large scale, during which the hybridized targeting DNAsight risk the unwound or detached issues.In the present work, we have developed a magnetic separation-

ased detection method for the sandwiched electroanalysis andiscrimination of DNAs using GOx with mineralized gold NPs (GOx-old) as the probe labels. As schematically illustrated in Scheme 1,erein, gold were doped into the protein matrix of GOx by theiomineralization route showing the greatly improved DETs andatalysis of GOx. Importantly, the resulted GOx-Gold compositesere confirmed with the double catalysis activities of enhancedOx catalysis and the peroxidase-like catalysis of mineralized gold.urthermore, after the hybridization of DNA targets, the capturend detection DNA probes would be linked through the oxidativeoupling reaction of their tyramine labels catalyzed by gold NPsf GOx-Gold. Meantime, GOx of GOx-Gold would catalyze the oxi-ization of glucose to produce H2O2 to facilitate the reductive goldrowth. The maximized and stable gold signals would be therebychieved because of the tyramine-mediated linking of DNA probes.

magnetic separation-based sandwiched detection method wasemonstrated by using GOx-Gold catalyst and tyramine linker ashe labels of DNA probes towards the sensitive electroanalysisnd discrimination of targeting Anthrax DNAs with the single-baseutation.

. Experimental section

.1. Reagents and apparatus

Glucose oxidase (GOx, EC 1.1.3.4, 200 U mg−1), hydro-en tetrachloroaurate (III) hydrate (HAuCl4·3H2O), Nafion5.0 wt.%) solution, horseradish peroxidase (HRP) (E.C. 1.11.1.7,00 U mg−1), tyramine, N-hydroxysuccinimide (NHS), N-thyl-N’-(3-dimethylaminopropyl) carbodiimide (EDC), andulfosuccinimimidyl 4-(N-maleimidomethyl) cyclohexane-1-arboxylate (Sulfo-SMCC) were purchased from Sigma-Aldrich.agnetic Dynabeads M-270 carboxylic acid (∼30 mg mL−1) was

btained from Invitrogen Dynal AS. The blood samples were kindlyrovided from the local hospital. The targeting DNAs were spiked

n blood to yield the DNA samples of different concentrations.

ll other chemicals were of analytical grade. Deionized wateras used throughout in the preparation of aqueous solutions

ike phosphate buffer solution (pH 7.2), and 2-(N-morpholine)thanesulfonate (MES) buffer (pH 5.0). Anthrax fetal factor DNA

tors B 261 (2018) 441–450

oligonucleotides of terminus-functionalized capture and detectionDNA probes, and matched and one-base mismatched DNAs weresynthesized by Shanghai Sangon (Shanghai), including:

(1) Capture probe: 5′-Thiol-(CH2)6-TAA CAA TAA TCC-(CH2)6-amine-3′;

(2) Detection probe: 5′-Amine-(CH2)6-ATC CTT ATC AAT ATT-(CH2)3-Thiol-3′;

(3) A-T match: 5′-GGA TTA TTG TTA AAT ATT GAT AAG GAT-3′;(4) A-G mismatch: 5′-GGA TGA TTG TTA AAT ATT GAT ATG GAT-3′;(5) A-C mismatch: 5′-GGA TCA TTG TTA AAT ATT GAT ATG GAT-3′;(6) A-A mismatched: 5′-GGA TAA TTG TTA AAT ATT GAT ATG GAT-

3′.

2.2. Synthesis and characterization of GOx-Gold

All glassware were first washed with nitro-hydrochloric acid(HNO3: HCl volume ratio = 1:3) (Caution: the mixture is a very cor-rosive oxidizing agent, which should be handled with great care.),and then rinsed with ethanol and ultrapure water. The synthesisof GOx-Gold was conducted by a modified procedure previouslyreported [32]. In a typical experiment, 2.0 mL aqueous HAuCl4solution (15 mM, 37 ◦C) was mixed with a 2.0-mL aliquot of GOxwith different protein concentrations (5.0, 10.0, 20.0, 30.0, 40.0,and 50.0 mg mL−1) under the vigorous stirring at 37 ◦C. An aliquotof NaOH (1.0 M) was then introduced into the mixture, and thereaction proceeded at 37 ◦C overnight. The resulting GOx-Goldcomposites were dialyzed in water overnight using the membranes(pore size of 1.8 nm or molecular weight of 20 KD). Finally, theywere stored at 4 ◦C for short-term usage, or dried by freezing in thesolid form for long-term usage.

Transmission electron microscopy (TEM) was operated on a FEITecnai TF-20 field-emission high-resolution transmission electronmicroscope at 200 Kv for imaging GOx-Gold products, which werepre-purified by the centrifuge. X-ray photoelectron spectroscopy(XPS) was applied to explore the oxidation states of Au in GOx-Gold. Moreover, a microplate reader (Tecan, Austria) and 96-wellplates were utilized in the tests for the peroxidase-like cataly-sis of GOx-Gold. The absorbance intensities of the reactions of3,3′,5,5′-tetramethylbenzidine and H2O2 (TMB-H2O2) were termi-nated with 2.0 M H2SO4 to be measured at 450 nm.

2.3. Electrochemical measurements using the GOx-Goldelectrodes

Glassy carbon electrodes (GCE, 3.0 mm in diameter) werecleaned following a common polishing procedure. An aliquot of5.0 �L GOx-Gold (5.0 mg mL−1 in GOx) that was pre-mixed withNafion (final concentration of 0.50 wt.%) was dropped onto the sur-face of a cleaned GCE to be dried overnight at 4 ◦C. Then, 5.0 �LNafion (0.25 wt.%) was casted and dried as a net to hold firmly theGOx-Gold onto the electrodes, resulting in the GOx-Gold modifiedelectrodes. The similar procedures were employed to fabricate theGOx-modified electrodes as the controls using native GOx with cor-responding enzyme concentrations. All of the resulting electrodeswere finally stored at 4 ◦C.

Electrochemical voltammetric measurements were carried outwith a CHI 760B electrochemical workstation (CH Instruments Co.,USA) using a conventional three-electrode cell. The GOx-Gold orGOx modified electrodes were used as the working electrodes. The

Pt wire and Ag/AgCl (3.0 M KCl) electrodes were used as the counterand reference electrodes, respectively. All measurement experi-ments were performed in the PBS that was piped with oxygen gasfor 10 min to facilitate the GOx-Gold-catalytic reactions.

H. Wang et al. / Sensors and Actuators B 261 (2018) 441–450 443

Scheme 1. Schematic illustration of the principle and procedure for the electrocatalysis and mutation discrimination of DNAs, including (A) the loading of DNA capturep ramins lectrog

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robe onto magnetic beads, the binding of DNA capture or detection probes with tyteps of the DNA hybridization, catalytic linking of DNA probes, gold growth, and elucose oxidization and tyramine coupling.

.4. The GOx-Gold-based detection and discrimination of DNAssing the screen-printed electrodes

The DNA capture probes derivatized with amines were firstound onto the carboxyl-functionalized magnetic beads accordingo the manufacture’s instruction. Basically, the EDC/NHS (0.10 M,H 5.0) agent was added to the pre-washed magnetic beads3.0 mg mL−1) and incubated with slow shake at room tempera-ure for 30 min. After being magnetically separated and washed, thectivated magnetic beads were mixed with the DNA capture probeolution (20 �M, pH 5.0) to be incubated at 4 ◦C overnight, andashed. The resulting magnetic beads-loaded DNA capture probesith terminal thiols were further labelled with tyramines by the

MCC bonding chemistry. Typically, tyramine (50 mM, pH 7.2) wasre-activated by sulfo-SMCC (20 mM, pH 7.2) for 30 min, and thendded to the above suspension to react for 1 h at room temperature.he resultants were washed and finally re-suspended in PBS bufferpH 7.2) to be stored at 4 ◦C. Moreover, following the similar proce-

ure above, the DNA detection or recognition probes were labelled

n turn with GOx-Gold and then tyramine by using EDC/NHS andulfo-SMCC, respectively. Here, the carboxyl-containing GOx-Gold5.0 mg mL−1 of GOx, pH 5.0) was first activated by EDC/NHS and

e, and the labelling of DNA detection probe with GOx-Gold; (B) the electrocatalysischemical measurements on SPE; and (C) the GOx-Gold catalyzed reactions for the

then bound with the amine-containing DNA probes (20 �M, pH5.0). The mixture was then treated with hydroxylamine hydrochlo-ride (100 mM) for 30 min, and purified by PD-10 desalting columns(Mr > 5000, GE Healthcare, UK). Furthermore, the resulting DNAdetection probes would be coupled with tyramine that was pre-treated with sulfo-SMCC as described above. After the purificationwith PD-10 columns, the so labelled DNA detection probes weresubsequently stored at 4 ◦C.

An aliquot of DNAs with matched or one-base mismatched baseat different concentrations was separately mixed with aliquots ofthe labelled DNA capture probes (1.0 mg mL−1 in magnetic beads)and DNA detection probes (1.0 mg mL−1 in GOx) in a plastic tube(1.0 mL). The hybridization reactions were conducted under theslow rotation at 4 ◦C for 2 h, followed by first washing in PBSbuffer (37 ◦C) and then the magnet-aided separation. Afterwards,an aliquot of glucose (10 mM) were introduced into the hybridiza-tion solution that was pre-piped with oxygen gas for 10 min andfurther incubated under oxygen for 20 min to yield H2O2 for the

catalytic oxidization of tyramine toward the linking of DNA probes.Furthermore, an aliquot of HAuCl4 solution (2.0 mM) containingcetyltrimethyl ammonium bromide (CTAB, 10 mM) was introducedinto the tube and incubated at room temperature for 20 min to

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44 H. Wang et al. / Sensors and

row gold NPs, followed by magnet-aided separation and washing. 100-�L aliquot of the diluted nitro-hydrochloric acid was thendded to the hybridized magnetic beads to dissolve the resultedold NPs into the solution. After mixing for 2 min, a 50-�L aliquot ofhe solution was drawn and then dropped onto the screen-printedlectrode (SPE, Pine Instrument company, USA) with three elec-rode configuration, which were pre-treated electrochemically byyclic voltammetry (CV) at a potential range of 0–1.5 V in PBS (pH.2). Subsequently, differential pulse voltammetry (DPV) measure-ents (the applied potential range of 0–1.4 V, increment of 10 mV,

nd amplitude of 50 mV) were performed after accumulation at.20 V for 60 s. Subsequently, the developed GOx-Gold modifiedlectrodes were employed for the detection of Anthrax DNAs spikedn blood with different concentrations according to the same proce-ure above. A baseline correction was performed for the resultingoltammograms using the CHI 760B software.

. Result and discussion

.1. Protocol of magnetic separation-based electroanalysis andiscrimination of DNAs using GOx-Gold

It is widely recognized that gold-labelled enzymes includingOx can allow for the biocatalytic gold growth [44–49]. In theresent work, magnetic separation-based sandwiched analysisrotocol for probing DNAs was proposed by separately using GOx-old catalyst and tyramine linker as the labels of DNA probes. Theain principle and procedure for the developed sandwiched elec-

roanalysis method using SPE electrodes is schematically illustratedn Scheme 1, in which Anthrax fetal factor DNA was chosen as aargeting DNA model. Herein, DNA capture probes were labelledeparately with magnetic beads and tyramine at two terminuses,o did the DNA detection probes labelled alternatively with theOx-Gold and tyramine (Scheme 1A). By following a magneticeparation-based sandwiched analysis way (Scheme 1B), the tar-et DNAs with matched bases (i.e., A–T) were hybridized with thewo probes on the magnetic carriers. Then, the linking of DNArobes would be realized by the H2O2-oxidized coupling of tyra-ine labels catalyzed by gold NPs of GOx-Gold. Meanwhile, the

lucose-induced gold growth would undergo through the oxida-ion of glucose catalyzed by GOx of GOx-Gold to produce gluconiccid and H2O2 [50]. Herein, the linking of DNA probes wouldid to achieve the maximized and stable gold signals. Scheme 1Cescribes the principle of the H2O2-oxidized coupling reactions ofyramine labels catalyzed by gold NPs of GOx-Gold. Accordingly,2O2 produced in the oxidization reaction of glucose (the upper)ould, on the one hand, spark the oxidative coupling of tyraminesatalyzed by peroxidase-like GOx-Gold (the lower). As a result, theatalytic linking of two tyramine-labelled DNA probes after theybridization with targeting DNAs onto the magnetic carriers coulde expected, named as the DNA linking shown in Scheme 1B. On thether hand, H2O2 could act as the reducing agent to reduce Au(III)ons into gold NPs that would be deposited onto the GOx-Goldabels, named as the gold growth, for the electrochemical output ofold signals. Remarkably, the yielded H2O2 might mostly surroundhe GOx-Gold catalysts with relatively high concentration so thatold NPs would be mostly yielded onto GOx-Gold [44,47]. Subse-uently, the electrochemical output of gold signals was conductedn the disposable SPE electrodes for DNA electroanalysis.

.2. Preparation and characterization of GOx-Gold with double

atalysis activities

It is well established that the active centers of enzymes likeRP and GOx are usually buried in the proteins or glycoproteins.

tors B 261 (2018) 441–450

Yet, they have evolved to possess molecular channels or pathwaysfrom the protein surface to the active sites [1,51]. According to theinformation of GOx structure [3], the active sites of flavin adeninedinucleotide are accessible only through a deep pocket that is fun-nel shaped with an opening of 10 × 10 Å at its surface, from whichthe distance to the active site is about 20–25 Å [52]. But the externalelectrons can hardly shuttle into the redox sites without an electro-chemical mediator of low molecular weight. Accordingly, we triedto in site mineralize gold into GOx, an enzyme with the catalysisunder alkaline environment, by using a protein-scaffold biominer-alization route [32]. Herein, gold precursors of Au (III) ions werefirst doped into the protein matrix of GOx and then reduced in situto form gold NPs encapsulated into or anchored at the enzyme pro-teins. Fig. 1A manifests the transmission electron microscopy (TEM)images of the resulted GOx-Gold composites prepared using differ-ent GOx concentrations, in which gold NPs were formed with thedistribution of particle sizes of about 0.80–20 nm in diameter. Also,the colors of the product solutions could change from yellow to redas the GOx protein concentrations decreased (Fig. 1B). Herein, onthe one hand, GOx could functionalize as the protein scaffolds andsurfactants for the formation of GOx-Gold by ensuring the size con-trol and high stability of gold NPs. On the other hand, Au(III) ionsmight penetrate into the catalytic centers of GOx protein mostly viathe entrance pathways of its deep pockets mentioned above, whichcould become more accessible when enzyme proteins conducteda slightly conformational denaturation under the basic environ-ment of the gold biominerization (i.e., pH 11). That is, the Au(III)ions could be thus reduced and further grow into gold NPs in situby the protein residues of GOx (e.g., tyrosine (Tyr) and cysteine).For example, Tyr residues can reduce metallic ions of Au (III) ionsthrough their phenolic groups with the reaction pH above its pKa(∼10) [32,36]. Furthermore, Fig. 1Ca manifests the TEM image of theGOx-Gold prepared typically using 20 mg mL−1 GOx protein, whichwas demonstrated afterwards as the optimal GOx concentration forpreparing the GOx-Gold with the highest peroxidise-like catalysis.One can note that the obtained GOx-Gold could have gold NPs withthe size of about 8.0 nm in diameter, in which gold could be pro-duced to anchor or spread throughout the whole protein moleculesof GOx. As clearly shown in the insert of Fig. 1Ca, herein, GOx pro-teins that otherwise are invisible in TEM image might be stainedby gold (red arrow) so as to be visualized to profile the ellipsoidalshape of dimeric GOx proteins with the overall dimensions of about70 × 55 × 80 Å [3], of which the residues responsible for the goldreduction are distributed throughout the enzyme proteins includ-ing their active centers (i.e., Tyr 68 and Tyr 515) [3]. Moreover, onecan note from Fig. 1Cb that gold NPs in GOx-Gold composites wouldfurther grow through the glucose-induced gold growth, showingthe gold particle size of about 17.0–18.0 nm in diameter, as appar-ently shown in the amplified view (insert). Herein, the gold growthwas achieved through the reduction of hydrogen peroxide yieldedin GOx-catalyzed reaction of glucose hydrolysis afterwards.

3.3. Colorimetric tests for the peroxidase-like catalysis activitiesof GOx-Gold composites

The peroxidase-like catalysis behavior of GOx-Gold compositeswas examined by the colorimetric tests by the catalytic TMB-H2O2chromogenic reaction (Fig. 2A), taking HRP for the comparison(insert). One can note that the catalysis performances of GOx-Goldcould be comparable to those of HRP. Moreover, the peroxidase-like catalysis of gold NPs of GOx-Gold could depend on the GOxconcentrations used for the gold formation, showing that the ones

with the highest catalysis should be prepared using 20 mg mL−1

of GOx protein. Accordingly, the optimized GOx-to-Au molar ratiowas calculated to be about 1/100, which was chosen in the sens-ing experiments afterwards. Since native GOx could not exhibit

H. Wang et al. / Sensors and Actuators B 261 (2018) 441–450 445

Fig. 1. GOx-Gold products prepared using GOx concentrations of 50, 40, 30, 20, 10, and 5.0 mg mL−1 corresponding to their (A) TEM images (a–f) and (B) photographs (No1 osites( 4.

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–6) under white light. (C) Comparison of typical TEM images for GOx-Gold compinsert: the magnification-amplified view) using 10 mM glucose and 2.0 mM HAuCl

he peroxidase-like catalysis (insert in Fig. 2A), such a uniqueeroxidase-like catalysis of GOx-Gold should be attributed to goldPs encapsulated. In addition, colorimetric measurements wereerformed to explore the environmental stability of GOx-GoldFig. 2B). Obviously, no significant change was observed in theatalysis performances of GOx-Gold composites even though theyere stored in water up to six months, thus confirming the high

nvironmental stability of GOx-Gold.

.4. GOx-Gold-based electroanalysis for glucose

Fig. 3A manifests the results of the GOx-Gold-based electro-atalysis for glucose, of which the insert one shows the CVs ofhe GOx-Gold modified electrodes at different sweep rates. In con-

rast to the one modified with native GOx, herein, the GOx-Gold

odified electrode can present much bigger and better definedair of redox peaks of flavin adenine dinucleotide in the bufferolution, with anodic and cathodic peak potentials at −0.440 V

prepared with 20 mg mL−1 GOx protein (a) before and (b) after the gold growth

and −0.498 V, respectively. The formal potential is calculated tobe about −0.469 V. It is compatible or better than those of theelectrodes fabricated elsewhere separately with gold NPs-labelledGOx (−0.434 V) [53], CNTs-GOx composites (−0.396 V or −0.403 V)[54,55], and GOx in the mesopous silica matrices (−0.436 V) [56]. Inaddition, X-ray photoelectron spectroscopy (XPS) analysis was con-ducted for gold NPs in GOx-Gold, showing the binding energies ofAu(0) oxidation state (Au 4f7/2, 84.0 eV; Au 4f5/2, 87.4 eV) (Fig. 3B).Accordingly, Au(0) in the GOx-Gold would help to improve the elec-trocatalysis of GOx-Gold in addition to the peroxidase-like catalysisabove. These data indicate that the GOx-Gold could obtain betterDET between the enzymatic active centers and the electrodes so asto allow for the direct electrocatalysis for glucose.

Furthermore, the typical amperometric responses of the GOx-

Gold modified electrode to glucose with the successively increasingconcentrations were examined. The corresponding current-timeresponses are shown in Fig. 4. One can see that well defined steady-state current responses were obtained at the GOx-Gold modified

446 H. Wang et al. / Sensors and Actuators B 261 (2018) 441–450

Fig. 2. (A) UV–vis absorbance intensities of the catalytic TMB-H2O2 reaction solu-tions versus GOx-Gold prepared using GOx of different concentrations, with theerror bars of five-replicate standard deviations. The insert shows the photographs ofthe resulting color solutions of TMB-H2O2 reactions catalyzed by GOx-Gold (bottomline) and native GOx with corresponding enzyme concentrations (top line) includ-ing HRP indicated by the arrow. (B) Investigations on the environmental stabilityoHu

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Fig. 3. (A) Comparison of electrochemical responses to glucose among the elec-trodes modified separately with blank, GOx, Gold-labelled GOx, and GOx-Gold at asweep rate of 50 mV s−1 in the N2-saturated PBS (pH 7.2). The insert shows the CVsof the GOx-Gold modified electrodes at different sweep rates of 10, 25, 50, 75, 100,and 200 mV s−1. (B) XPS spectra of GOx-Gold under the freeze drying condition.

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f GOx-Gold stored in water over different time intervals, where the catalytic TMB-2O2 reactions were performed to monitor the catalysis activities of the GOx-Goldsing 1.6 mM H2O2 and 0.62 mM TMB.

lectrodes, which currents increased stepwise with the succes-ive additions of glucose. The calibration plots are shown in thensert of Fig. 4. The steady-state currents at the GOx-Gold modi-ed electrodes were proportional to glucose in the concentrationange of 0.50 mM–3.5 mM, with the detection limit evaluated toe 0.125 mM. The response time of the developed electrodes waseasured to be less than 8 s, reaching 95% of the maximum change

n response to a step injection of glucose. Therefore, the GOx-Goldodified electrodes can be tailored for rapid and direct electroanal-

sis for glucose.

.5. GOx-Gold catalyzed gold growth triggered by glucose

The catalysis abilities of GOx-Gold for catalyzing the glucose-riggered gold growth, which would facilitate the electrochemicalutput of gold signals for the electroanalysis of targeting DNAs,ere demonstrated optically and electrochemically (Fig. 5). Fig. 5A

hows that the photographs of reaction products of the glucose-nduced gold growth catalyzed by the GOx-Gold composites.ccordingly, the colors of the reaction products could change from

ight yellow to dark purple depending on the GOx-Gold compositesrepared using different dosages of GOx protein, taking the nativeOx-catalyzed one as the control. The above results indicate that

he glucose-induced gold growth could be attributed to gold NPsf GOx-Gold labelled on DNA detection probes that would act ashe catalysts as well as gold seeds, where the H2O2 yielded fromhe glucose hydrolysis catalyzed by the GOx-Gold labels would

successive additions of glucose at the 0.125 mM step with a sweep rate of 50 mV s−1

in PBS (pH 7.2) using an applied potential of 0.40 V versus Ag/AgCl. The insert showsthe calibration curve of the currents versus different glucose concentrations.

H. Wang et al. / Sensors and Actuators B 261 (2018) 441–450 447

Fig. 5. (A) Photographs of the glucose-induced gold growth catalyzed by GOx-Goldprepared with GOx concentrations of 10, 20, 30, 40, and 50 mg mL−1 (No 1–5), takingnative GOx as the control. (B) Comparison of the gold responses between the sand-wiched DNA detections with and without the gold growth (the applied potentialrange of 0–1.4 V versus Ag/AgCl, increment of 10 mV, and amplitude of 50 mV). TheiA

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Fig. 6. Comparison of electrochemical DPV responses between the DNA detectionswith and without the tyramine-mediated linking of DNA probes in the glucose-induced gold growth catalyzed by GOx-Gold using 10 mM glucose and 2.0 mMHAuCl4 The five replicated tests were conducted for the DNA electrodes showingthe stability of the DPV responses to the gold so deposited. The applied potentialrange of 0–1.4 V versus Ag/AgCl, increment of 10 mV, and amplitude of 50 mV. The

nsert shows the typical CV characteristics of gold response of the DNA electrode.ll results were the average values of five replicates.

erve as the reducing agent to turn Au(III) ions into gold NPs to beeposited onto the GOx-Gold labels. More importantly, the devel-ped DNA biosensor based on the GOx-Gold-catalytic gold growthould achieve much larger electrochemical outputs of sharp goldignals, which are much larger than those of the ones without theold growth (Fig. 5B), thus promising higher detection sensitivityn sensing DNAs. It is worthy noting that glucose itself may alsoeduce Au3+ ions into gold in the testing droplets, which howeverould be removed from the electrode surface during the cleaning

tep.

.6. The oxidative coupling of tyramine labels catalyzed byOx-Gold

It is well established that peroxidases like HRP can catalyze theoupling reactions of a number of phenol and aniline derivativesith H2O2 as the oxidant [57], typically as the oxidative coupling

f tyramines [58,59]. Moreover, the traditional sandwiched DNAetections were mostly performed without the linking of DNArobes including the ones with the catalytic gold growth [60,61].s a result, the yielded signals might be commonly trapped by the

imited signal amplification leading to the limited detection sen-itivities of DNA biosensors. Alternatively, herein, tyramine wasmployed to separately label the capture and detection DNA probeso mediate the linking of DNA probes after the target hybridiza-ion, where the gold NPs of GOx-Gold would additionally act as the

eroxidase-like catalysts for catalyzing the oxidative coupling ofyramine labelled on two DNA probes. The glucose-induced goldrowth catalyzed by GOx-Gold could be thus maximized together

top photographs are the TMB-H2O2 reaction solutions containing magnetic beadswithout (left) and with (right, three replicated tests) the tyramine-mediated linkingof DNA probes after the DNA hybridizations.

with the high stability on the magnetic carriers to expect the greatlyamplified signals of gold growth.

In order to confirm the tyramine-mediated linking of DNAprobes, the resulting magnetic beads were magnetically separatedafter the linking of DNA probes (only adding glucose withoutHAuCl4) and further added into the TMB-H2O2 reaction solutions,taking the ones without the linking of DNA probes as the con-trol. The photographs of the reaction solutions are comparablyshown on the top of Fig. 6. As expected, blue product solutionswere observed only for the magnetic beads with the tyramine-mediated linking of DNA probes, in which Gold NPs of GOx-Goldso anchored would catalyze the TMB-H2O2 reactions. Hence, thetyramine-mediated linking of DNA probes could be realized forthe DNA detections based on the catalytic gold growth afterwards.Moreover, a comparison of electrochemical gold signals was con-ducted between the DNA detections with and without the linkingof two DNA probes labelled with tyramines, of which five replicatedtests for targeting DNAs were conducted to examine the stabilityof electrochemical gold signals (Fig. 6). One can find that the devel-oped DNA biosensor using tyramine as the labels of DNA probescould achieve much more stable and bigger gold signals, which isabout three times larger than that of the one without the use of tyra-mine labels. The above results confirm that the tyramine-mediatedlinking of DNA probes can promise the maximized amplification ofthe gold signals in the GOx-Gold catalytic reactions toward highlysensitive DNA eletroanalysis as demonstrated afterwards.

3.7. The GOx-Gold-based sandwiched electroanalysis for AnthraxDNAs

The electroanalysis performances of the developed sandwichedmethod were investigated for Anthrax DNAs spiked in blood withdifferent concentrations using the GOx-Gold modified electrodes

448 H. Wang et al. / Sensors and Actua

0.2 0.4 0.6 0.8 1.0

0.0

2.0

4.0

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8.0A-T matched DN A A-C mismatched DNA A-G mismatched DNA A-A mismatched DNA Control (blank)

1E-7 1E-6 1E-5 1E-4 1E-3 0.010.0

3.0

6.0

9.0

Log[DNA ] (nM)

Fig. 7. (A) The electrochemical response-concentration calibration curve plottedusing different concentrations of DNA spiked in blood on a semi-log scale, withthe error bars of five replicates showing the standard deviations. The insert showsthe in-situ DPV responses to the target DNAs. (B) Comparison of electrochemicalresponses for typical DNA discrimination between the DNAs with matched andone-base mismatched sequences using an applied potential range of 0–1.4 V versusAg/AgCl, increment of 10 mV, and amplitude of 50 mV.

Table 1Comparison of detection performances among the different analysis methods forDNAs.

Analysis methods Linear ranges Detection limit

Fluorescence Analysis [62] 0.20 nM–64 nM 0.20 nMElectrochemical Analysis [63] 0.10 fM–20 fM 0.08 fM

(bIclisDGtdtd

dations of China (Nos. 21675099 and 21375075), the National

Hybridization Chain Reaction [64] 0.20 pM–0.50 �M 1.00 fMThis Work 0.20 fM–6.4 pM 0.10 fM

Fig. 7). The response-concentration calibration curve was obtainedy plotting the DNA concentrations on a semi-log scale (Fig. 7A).t is found that the targeting DNAs could be detected in the con-entrations ranging from 0.20 fM to 6.40 pM, with the detectionimit of ∼0.10 fM (by 3� rule), of which the in-situ electrochem-cal responses could depend on different DNA concentrations ashown in the insert of Fig. 7A. Moreover, compared to some currentNA analysis methods reported previously [62–64], the developedOx-Gold-based sandwiched electroanalysis strategy can present

he better or compatible detection performances in terms of linearetection range and detection limit (Table 1). Also, high detec-ion reproducibility of the developed electroanalysis method wasemonstrated showing no significant change in the responses to

tors B 261 (2018) 441–450

the same level of DNA target within six repeated tests (data notshown). Moreover, Fig. 7B shows a comparison of electrochemicalresponses between the base matched and one-base mismatchedDNAs. One can find that when the DNA sequences with mismatchedbases (i.e., A–G) were analyzed, the formed DNA hybrids could bereadily dehybridized and detached from magnetic beads depend-ing on the extent of the dehybrization once washed at 37 ◦C thatis beyond the melting temperature of the base-mismatched DNAsequences. Herein, lower catalytic linking of tyramine-labelledDNA probes and then less glucose-induced gold growth wouldoccur on the magnetic carriers. As a result, an apparent difference ofgold signals was comparably observed between the matched andmismatched DNAs, thus confirming the feasibility for the sensi-tive discrimination of DNAs with single-base mutation. Therefore,the developed magnetic separation-based DNA biosensor usingdouble-catalysis GOx-Gold can enable the highly sensitive elec-troanalysis and selective discrimination of DNAs with single-basemutation.

4. Conclusion

A magnetic separation-based detection method has been suc-cessfully developed for the sandwiched electroanalysis and thebase-mutation discrimination of Anthrax DNAs by using bi-catalytic GOx-Gold catalyst and tyramine linker separately as thelabels for DNA probes, in which the catalytic linking of tyramine-labelled DNA probes and gold growth would be involved. Thedeveloped DNA electroanalysis method can present some uniqueadvantages over the traditional ones especially these by way ofthe sandwiched DNA hybridization and the enzyme catalysis-basedsignal amplification routes. Moreover, comparing to native GOxthat are modified or labelled directly with electronic relays or con-ductive nanomaterials with limited DET and catalysis, here, GOxwas alternatively doped with gold by the in-situ biomineralizationroute, showing the greatly enhanced intrinsic catalysis and elec-trocatalysis of GOx. Especially, the prepared GOx-Gold compositescould thus feature the double catalysis activities of peroxidase-likegold and GOx catalysis activities. The colorimetric and electroan-alytic results indicate that after the DNA hybridization, the DNAcapture and detection probes could be linked through the cou-pling of tyramines labels catalyzed by the gold NPs of GOx-Gold.Meantime, GOx of GOx-Gold could catalyze the oxidization of glu-cose to produce H2O2 to facilitate the catalytic gold growth. It wasconfirmed that the catalytic linking of two tyramine-labelled DNAprobes could help to achieve the maximized and more stable goldsignals towards the greatly improved DNA detection performances.The so developed sandwiched detection method can facilitate theultrasensitive electroanalysis of Anthrax DNAs spiked in blood aswell as the discrimination of targeting DNAs with single-base muta-tion. Also, equipped with the magnetic separation, it can allow forthe analysis of the targets in the complicated media (i.e., blood).Importantly, this one-pot and simple gold biomineralization routemay open a new door towards the synthesis of various DET-improved enzymes with dramatically enhanced intrinsic catalysisand electrocatalysis. Yet, it remains unclear at the molecular levelhow gold NPs can reductively grow inside the enzymes. Furthermolecular-level insights and new applications of GOx-Gold prod-ucts are under way in our group.

Acknowledgements

This work is supported by the National Natural Science Foun-

Natural Science Foundation of Shandong Province (ZR2014BM025and ZR2015PB014), and the Taishan Scholar Foundation of Shan-dong Province, P. R. China.

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Shuyun Zhu received her Ph.D. from Changchun Institute of Applied Chemistry,Chinese Academy of Sciences, China, in 2012, and now is working at Qufu NormalUniversity as an associate professor in Chemistry. Her research interests mainlyinclude Biosensors and Medical Detectors R&D.

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iographies

ua Wang received his Ph.D from Hunan University, China, in 2004, and now isorking at Qufu Normal University as a distinguished professor in Chemistry. His

esearch interests mainly include Chemo/Biosensors, Advanced Functional Materi-ls, and Organic Synthesis.

iyan Zhang received her bachelor degree in Chemistry Education from the Qufuormal University in China, in 2014, and now is a postgraduate student in Analytical

tors B 261 (2018) 441–450

Chemistry at the same university. Her research interests include Biosensors andMedical Detectors R&D.

Yao Jiang received his bachelor degree in Chemical Engineering and Technologyfrom the Qufu Normal University, China, in 2014, and now is a postgraduate studentin Analytical Chemistry at the College of Chemistry and Chemical Engineering inthe same university. His research interests mainly include Biosensors and MedicalDetectors R&D.

Lijun Chen received her Medicine degree from Hunan University of TraditionalChinese Medicine, China, in 1991, and now is working at the Hospital of Qufu Nor-mal University as a doctor-in-charge. Her research interests mainly include ClinicalBioanalysis and Diagnostics.

Zhiqiang Duan received his Ph.D. from Nanjing University, China, in 2014, and nowis working at Qufu Normal University as a post-doctoral staff in Chemistry. Hisresearch interests mainly include Biosensors, Medical Detectors R&D, and OrganicAnalytical Chemistry.

Xiaoxia Lv received her Ph.D. from Qingdao Institute of Bioenergy and Process Sci-ence, Chinese Academy of Sciences, China, in 2014, and now is working at QufuNormal University as an associate professor in Chemistry. Her research interestsmainly include Biosensors and Medical Detectors R&D.