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Sensors and Actuators B 192 (2014) 157– 163

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l h om epage: www.elsev ier .com/ locat e/snb

mplified detection of T4 polynucleotide kinase activity based on a-exonuclease cleavage-induced DNAzyme releasing strategy

hufeng Liua, Jingjing Minga, Ying Lina, Chunfeng Wanga,ao Liua, Chuanbin Chenga, Feng Lib,∗

College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People’s Republic of ChinaCollege of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, People’s Republic of China

r t i c l e i n f o

rticle history:eceived 16 August 2013eceived in revised form 15 October 2013ccepted 22 October 2013vailable online 30 October 2013

eywords:4 polynucleotide kinase-Exonuclease

a b s t r a c t

The T4 polynucleotide kinase (T4 PNK) plays an essential role in the cellular responses to nucleic acidstrand damage. Herein, a simple and sensitive fluorescence approach for monitoring T4 PNK activity wasproposed based on a �-exonuclease (�-exo) cleavage-induced DNAzyme releasing strategy. A hairpin-shaped DNA probe that contains the sequence of 8-17 DNAzyme as a built-in suppressed catalytic unitwas designed. After phosphorylation by T4 PNK followed with the immediate �-exonuclease (�-exo)cleavage, the 8-17 DNAzyme unit was successfully released and used for the cyclic cleavage toward themolecular beacon substrate, resulting in an evident fluorescence signal enhancement. With the currentlydeveloped �-exo cleavage reaction and DNAzyme-based platform, the amplified detection of T4 PNK with

−1

luorescence sensorNAzyme

a low detection limit of 0.005 U mL could be achieved. Furthermore, the inhibition effects of adenosinediphosphate, ammonium sulfate, and sodium hydrogen phosphate have been evaluated. The developed�-exo cleavage-induced DNAzyme releasing strategy opens a promising avenue for monitoring activityand inhibition of nucleotide kinase, and should be also easily extended for the sensitive detection towardmany other nucleic acid enzymes and may find widespread applications in biological process researches,

diag

drug discovery, and clinic

. Introduction

The 5′-polynucleotide kinase (PNK) that can catalyze the phos-horylation of nucleic acids with 5′-hydroxyl termini plays a crucialole in regulation of many important cellular events, particularly inhe cellular responses to nucleic acid strand damage and interrup-ion [1–5]. It has been well known that the DNA lesions causedy some exogenous and endogenous agents, for instance, chemi-al substances [6], ionizing radiation [7], as well as nucleases [8],ften possesses 5′-hydroxyl termini. This would lead to the fail-re of 5′-phosphate termini-dependent repair processes for nucleiccids and result in serious consequences, even some lethal diseases.ccordingly, phosphorylation at the 5′-termini is an inevitableccurrence before broken strands rejoining can be finished. T4olynucleotide kinase (T4 PNK), first discovered in 1965 [9], is theounding member of a family of 5′-kinase. It can catalyze the trans-er of the �-phosphate residue of ATP to the 5′-hydroxyl group of

ucleic acids, and has been widely used in detection of DNA adducts10] and in repair of nucleic acid lesions [11,12]. Blockage of DNAhosphorylation or aberrant T4 PNK activity may be closely related

∗ Corresponding author. Tel.: +86 532 86080213; fax: +86 532 86080213.E-mail address: lifeng@qust.edu.cn (F. Li).

925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.snb.2013.10.101

nostics.© 2013 Elsevier B.V. All rights reserved.

to some serious human disorders for example Bloom’s syndrome,Werner syndrome, and Rothmund–Thomson syndrome [13]. Untilnow, various approaches for the determination of DNA phospho-rylation have been developed. The traditional T4 PNK assays areperformed with radical isotope 32P-labeling, polyacrylamide gelelectrophoresis (PAGE), and autoradiography [9,14–18]. However,these methods are usually confronted with complicated instru-ments, sophisticated operating procedures, and might be harmfulto human health. Recently, fluorescence method based on molec-ular beacon (MB) or nanobeacon has emerged as an increasinglypopular tool for DNA phosphorylation assay owing to its distinctiveadvantages such as convenient, high-throughput, cost-effective,and sensitive. For example, Tang et al. described a novel fluores-cence assay for sensitive monitoring of the phosphorylation processusing a MB based on a ‘phosphorylation-ligation’ coupled enzymereaction strategy [4]. Song et al. proposed a novel fluorescenceapproach for real-time monitoring of the activity and kinetics ofT4 PNK by using a singly fluorophore-labeled DNA-hairpin probecoupled with �-exonuclease (�-exo) cleavage reaction [19]. Fur-thermore, Wu et al. and Lin et al., respectively, successfully screened

DNA phosphorylation process by using grapheme oxide as a superquencher [20,21]. Although these fluorescence methods have madegreat advances toward the DNA phosphorylation assay, it should benoted that the further improvement of the analytical performances,

158 S. Liu et al. / Sensors and Actuators B 192 (2014) 157– 163

for the

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(25 mM HEPES, 100 mM NaCl, pH 7.0) were added into the sam-ple solution. After incubation for 30 min, the fluorescence intensitywas then recorded.

Fig. 2. Fluorescence emission spectra of the sensing system in the presence of dif-ferent concentrations of T4 PNK (a: 0; b: 0.2; c: 10 U mL−1). The reaction buffer alsocontains 150 nM hairpin DNA probe, 1 mM ATP and 10 units �-exo. The curve d isfor the control experiment using 150 nM phosphorylated hairpin probe instead of

Fig. 1. Schematic illustration

articularly sensitivity, is still in high demand. On the basis of theseesearch efforts, by the integration of appropriate signal amplifi-ation strategy, the sensitivity improvement for T4 PNK activityetection should be reasonably conceivable.

Signal amplification is an efficient way to construct biosensingystems with high sensitivity. The signal amplification strategieseveloped to improve the sensitivity of sensing event are usu-lly operated by the use of protein enzyme such as endonucleasend exonuclease, etc. as biocatalysts [22–29]. Compared with pro-ein enzymes, DNAzymes are a variety of catalytic DNA sequenceselected by SELEX that show high catalytic hydrolytic cleavagectivities toward specific substrates, while they are more stablehan enzymes, and can be denatured and renatured many timesithout losing their catalytic activities toward substrates [30]. Thisnique advantage makes DNAzymes ideal biocatalysts for ampli-ed sensing applications toward various targets, such as metal

ons, DNA, miRNAs, enzyme activity, small molecules and so on31–38]. As a typical example of DNAzymes, 8-17 DNAzyme haseen extensively employed for amplified detection of a variety ofargets [33–36].

Herein, a simple, rapid and amplified detection toward T4NK activity was proposed based on a �-exo cleavage-inducedNAzyme releasing strategy. The active 8-17 DNAzyme unit coulde successfully released from the designed hairpin DNA probefter 5′-phosphorylation by T4 PNK followed with the imme-iate �-exonuclease (�-exo) cleavage, and further used for theyclic cleavage toward the ribonucleotide-containing MB substrate,esulting in an evident fluorescence signal enhancement toward4 PNK activity detection. To the best of our knowledge, thishould be the first application of DNAzyme releasing strategy forignal amplification in T4 PNK activity detection. It would be aery beneficial supplementary to the field of nucleotide kinaseetection.

. Experimental

.1. Materials and chemicals

T4 polynucleotide kinase (10 units/�L) and �-exonuclease5 units/�L) were purchased from New England Biolabs (NEB,.K.). Adenosine 5′-triphosphate (ATP), adenosine diphos-hate (ADP) and dithiothreitol (DTT) were purchased fromangon Biotech Co., Ltd. (Shanghai, China). All other chemicalsere obtained from Shanghai Chemical Reagents (Shanghai,hina) and used without further purification. Milli-Q waterresistance >18 M� cm) was used in all experiments. The DNA

ligonucleotides used in this work were synthesized and puri-ed by Takara Biotechnology Co., Ltd. (Dalian, China), andheir sequences are listed as follows: Hairpin DNA probe: 5′-CCAACTATTGTTCATCTCTTCTCCGAGCCGGTCGAAATAGTTGGT-3′;

detection of T4 PNK activity.

Phosphorylated hairpin probe: 5′-PO4-ACCAACTATTGTTCATCT-CTTCTCCGAGCCGGTCGAAATAGTTGGT-3′; 8-17 DNAzyme probe:5′-GTTCATCTCTTCTCCGAGCCGGTCGAAATAGTTGGT-3′; MB: 5′-FAM-CCACCACATTCAAATTCACCAACTATrAGGAAGAGATGTTACG-AGGCGGTGGTGG-BHQ-3′; All the DNA were used as provided anddiluted in 10 mM Tris–HCl buffer (pH 7.5) to give the stock solutionof 1 �M.

2.2. Procedures for T4 PNK activity detection

In a typical phosphorylation assay, 100 �L of reaction buffer(70 mM Tris–HCl, 10 mM MgCl2, 5 mM DTT, pH 7.5) containing150 nM hairpin probe DNA, 1 mM ATP, 10 units of �-exo, and a cer-tain amount of T4 PNK was incubated at 37 ◦C for 30 min. After that,the resulting mixture was heated to 75 ◦C for 20 min to deactivatethe T4 PNK and �-exo, and then allowed to cool to room tem-perature. After T4 PNK phosphorylation and �-exo cleavage, 30 �Lof 1 �M MB, 10 �L of 1.5 mM Pb2+ and 160 �L of buffer solution

unphosphorylated hairpin probe with addition of 10 units of �-exo and no T4 PNK.The curve e is obtained by using 150 nM synthesized 8-17 DNAzyme fragment with-out addition of T4 PNK and �-exo. The inset in Fig. 2 shows the gel electrophoresisanalysis of 1 �M hairpin DNA probe without (1) and with (2) addition of 10 U mL−1

T4 PNK in the presence of 10 units of �-exo.

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.3. Kinase inhibitor evaluation

To evaluate the effects of inhibitors on the T4 PNK-catalyzedhosphorylation process, several kinds of inhibitors, including ADP0.1–7.5 mM), (NH4)2SO4 (5–30 mM), and Na2HPO4 (10–50 mM),ere also contained in the reaction buffer, respectively. After the

ddition of 150 nM hairpin probe DNA, 1 mM ATP, 10 units of �-xo, and 1 unit of T4 PNK, the reaction was performed at 37 ◦C for0 min. The following procedures were similar as above.

.4. Instruments

Fluorescence spectra were measured with an F-4600 fluores-ence spectrophotometer with a scan rate at 1200 nm/min. Thexcitation wavelength was set at 480 nm, and the 24 photomul-iplier tube voltage was 700 V. The slits for excitation and emissionere set at 10 nm/10 nm. The gel electrophoresis experiments wereerformed by using a JY-SCZ2+ Electrophoresis Cell (Beijing Junyi-

ongfang Electrophoresis Equipment Co., LTD) equipped with a JY00C Electrophoresis Power Supply (Beijing Junyi-Dongfang Elec-rophoresis Equipment Co., LTD). In the gel electrophoresis assay,ach prepared sample (10 �L) was put on a 20% nondenaturing

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ig. 3. (A) The effect of the molar ratio of MB to hairpin probe on the fluorescence resp0 U mL−1 T4 PNK; (B) Optimization of the reaction time in the presence (black curve) an

n the reaction buffer were 1 mM and 10 units, respectively; (C) Optimization of �-exo

mM and 10 U mL−1, respectively. The reaction time was 30 min; (D) Optimization of ATP0 U mL−1 and 10 units, respectively. The reaction time was 30 min. (For interpretation of tf this article.)

rs B 192 (2014) 157– 163 159

polyacrylamide gel to separate the related substances. The elec-trophoresis was carried out in 1 × TBE (pH = 8.3) at 160 V constantvoltage for about 180 min at room temperature. The gels werescanned using the Omega 16ic Gel imaging system (ULTRA-LUM,USA).

3. Results and discussion

3.1. Design of strategy

The developed strategy for investigating T4 PNK activity wasschematized in Fig. 1. The 8-17 DNAzyme was chosen as the cat-alytic unit for the amplified detection of T4 PNK activity based on itshighly catalytic activity and the ability to expand its functionality byadopting either Pb2+ or Zn2+ as cofactors. Herein, a hairpin-shapedDNA probe was designed, which consists of the sequence of 8-17DNAzyme at the 3′ end (green fragment) that is caged in the duplexstructure of the stem by an interfering sequence at 5′ terminus (red

fragment). In such case, the 8-17 DNAzyme activity is suppressedbecause it cannot form the active conformation to hybridize withthe MB substrate. Also, this hairpin DNA probe with the hydroxylgroup at 5′-terminus can not be effectively digested by �-exo, which

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onse of the sensing system in the absence (red bar) and presence (green bar) ofd absence (red curve) of 10 U mL−1 T4 PNK. The concentrations of ATP and �-exo

concentration. The concentrations of ATP and T4 PNK in the reaction buffer were concentration. The concentrations of T4 PNK and �-exo in the reaction buffer werehe references to color in this figure legend, the reader is referred to the web version

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between ATP and DNA to T4 PNK, whose binding sites for DNA werepartially blocked by ATP [4,20]. Thus, the optimal concentration ofATP was chosen to be 1 mM.

Fig. 4. (A) Fluorescence emission spectra of the sensing system on exposure to dif-ferent concentration of T4 PNK (from bottom to top, 0, 0.005, 0.01, 0.02, 0.05, 0.1,

60 S. Liu et al. / Sensors and A

s a highly processive exonuclease from 5′ to 3′ end that prefer-ntially degrades double stranded DNA with a phosphate moietyt 5′ terminus to yield mononucleotides and single-stranded DNA,roviding a low background signal for the sensing system [39–41].evertheless, when both the T4 PNK and �-exo are present in

he test solution, the T4 PNK will catalyze the phosphorylationeaction toward the hydroxyl group at 5′-terminus of the hairpin-haped probe to yield the 5′-phosphorylated hairpin probe [42].ubsequently, the phosphorylated hairpin probe can be immedi-tely cleaved by �-exo, resulting in the release of 8-17 DNAzymeequence for the recovery of cleavage activity toward its substrate.he ribonucleotide-containing MB substrate for the 8-17 DNAzymes modified with a 6-carboxyfluorescein dye (FAM) as fluorophoret its 5′ terminus and a black hole quencher (BHQ) at its 3′-terminus.he ribonucleotide position in the MB substrate is marked in redolor. The liberated 8-17 DNAzyme can then hybridize with theB substrate and its catalytic cleavage activity toward MB sub-

trate is activated upon the addition of the cofactor of Pb2+ ions43–45], causing the quenched MB fluorophore/quencher pair toe separated, thereby producing a dramatic increase of fluorescentignal. Eventually, each released 8-17 DNAzyme can undergo manyycles to trigger the cleavage of many MB substrates, providing anmplified detection signal for T4 PNK activity.

.2. Monitoring of the T4 PNK-catalyzed phosphorylation

To demonstrate the feasibility of our method for monitoring4 PNK activity, a 5′-phosphorylated hairpin-shaped probe waseforehand synthesized and used for comparison. As shown inig. 2, in the presence of only �-exo but not T4 PNK, the phos-horylated hairpin probe catalyzed by �-exo cleavage showed aramatic enhancement of fluorescence, while the unphosphory-

ated hairpin DNA probe showed only little fluorescence change inhe reaction mixture. This is because of the preference of �-exo for

phosphate moiety at 5′ terminus of dsDNA. In the absence of the′-phosphate, �-exo shows only low activity due to the formationf inert enzyme-substrate complexes. However, when the hairpinNA probe reacted with both T4 PNK and �-exo, a much higheruorescence was observed. It could be also found that the fluo-escence intensity increased with the increase of addition amountf T4 PNK. The fluorescence intensity of the sensing system cat-lyzed by 10 U mL−1 T4 PNK and �-exo was about 10 times higherhan that of the system treated by �-exo only. Furthermore, the-17 DNAzyme fragment had also been synthesized and used foromparison. It could be found that the sole addition of this syn-hesized fragment can induce an evident fluorescence response inhe absence of T4 PNK and �-exo. This indirectly reflected thathe cleaved 8-17 DNAzyme fragment from hairpin-shaped probeatalyzed by T4 PNK coupled with �-exo contributed to the fluores-ence signal readout after its specific interaction with MB substrate.he above experiments strongly verified the feasibility of our devel-ped method for the monitoring of T4 PNK activity. To furtherlarify the digestion of the hairpin-shaped DNA probe by T4 PNKn our system, gel electrophoresis was also performed. Follow-ng addition of T4 PNK and �-exo, an evident cleavage fragmentppeared on the gel as expected (inset in Fig. 2). This also evidencedhat the hairpin-shaped DNA probe could be phosphorylated by T4NK and further be digested by �-exo, resulting in the release ofctive 8-17 DNAzyme fragment.

.3. Optimization of assay conditions

In order to achieve the system’s best sensing performance, theffect of molar ratio of MB to hairpin probe on the fluorescenceesponse of the sensing system was firstly investigated. Experi-ental results showed that a molar ratio of MB to hairpin probe

rs B 192 (2014) 157– 163

at 2 could provide the best sensing performance for the detectionof 10 U mL−1 T4 PNK (Fig. 3A). Although higher molar ratio couldresult in an increased fluorescence response, the background sig-nal in the absence of T4 PNK was unsatisfactory. Thus, a molar ratioof MB to hairpin probe at 2: 1 was therefore chosen for furtherexperiments. The assay conditions including the reaction time, theamounts of �-exo and ATP were further optimized. The reactiontime was a crucial parameter for the T4 PNK-catalyzed phospho-rylation and the coupled �-exo cleavage process. An excess ofincubation time would make the fluorescence signals unreliable as�-exo would also hydrolyze dsDNA with 5′-hydroxyl slowly [20]. Itcould be found that the fluorescence intensity increased graduallywith the increase of the reaction time and then reached equilibriumin 30 min, suggesting the complete phosphorylation and cleavageprocess (Fig. 3B). It should be noted that only very weak fluores-cence intensity increase was observed in the absence of T4 PNK. Thefluorescence intensity was also found to increase with the amountof �-exo and almost reached the saturation value at an amount of 10units of �-exo (Fig. 3C). Thus, the optimization time and the amountof �-exo were chosen to be 30 min and 10 units. The effect of ATPconcentration on kinase activity was also studied and shown inFig. 3D. As could be seen, the fluorescence intensity increased withthe increasing concentrations of ATP and then reached its maxi-mum at the ATP concentration of 1 mM. When the concentration ofATP was higher than 1 mM, the fluorescence signal decreased again.This inhibition effect was a consequence of the competitive binding

0.2, 0.5, 1, 2, 5, 10 U mL−1) in reaction buffer; (B) Calibration curve of the sensingsystem for the T4 PNK activity. The inset in Fig. 4B shows the linear relationshipbetween F/F0 value and T4 PNK concentration from 0.005 to 0.2 U mL−1. F and F0

are the fluorescence intensity of the sensor in the presence and absence of T4 PNK,respectively.

S. Liu et al. / Sensors and Actuators B 192 (2014) 157– 163 161

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ig. 5. Inhibition effects of (A) ADP, (B) (NH4)2SO4, and (C) Na2HPO4 on phosphoryl0 U mL−1 T4 PNK, 1 mM ATP, and 10 units �-exo.

.4. Fluorescence measurement of T4 PNK activity

The activity of T4 PNK was then investigated under the opti-ized conditions. Fig. 4A shows the fluorescence responses for

ifferent T4 PNK concentrations ranged from 0.005 to 10 U mL−1.t was observed that the fluorescence intensity gradually increased

ith increasing T4 PNK concentration. This was in accordance withhe fact that under higher concentrations of T4 PNK, more activated-17 DNAzymes were released, and catalyzed the cleavage of moreB substrates to induce a gradual increase of fluorescence inten-

ity. Fig. 4B depicts the calibration curve of the sensing system.he F/F0 value was linearly increased with the T4 PNK concentra-ion in the range from 0.005 to 0.2 U mL−1. The directly measuredetection limit for T4 PNK was obtained at 0.005 U mL−1, which wasuperior to or comparative with the reported methods (see Table1 in Appendices A and B). The high sensitivity of current strat-gy could be ascribed to cyclic cleavage of released 8-17 DNAzymeoward its substrate for amplified fluorescence signal.

.5. T4 PNK activity inhibition evaluation

The inhibition effects of adenosine diphosphate (ADP), ammo-ium sulfate and sodium hydrogen phosphate on phosphorylationad been investigated through the proposed detection platform.hese chemicals are considered to have no inhibition effect onhe activity of �-exo and can be used as inhibitors for T4 PNK4,20]. Increasing concentrations of ADP resulted in a decrease ofhe fluorescence intensity (Fig. 5A), indicating the weakening ofNA phosphorylation. The addition of 1.0 mM ADP had caused anbout 50% decrease in DNA phosphorylation. This was owing to theeversible phosphorylation reaction when ADP and 5′-phosphorylucleic acids existed in the reaction buffer simultaneously. Theossible salt effects on T4 PNK activity were also checked by

ddition of ammonium sulfate or sodium hydrogen phosphate. Itould be found from Fig. 5B and C that the fluorescence intensityecreased with the increasing concentration of ammonium sulfatend sodium hydrogen phosphate, indicating the inhibition effect

The assays were carried out in the reaction buffer containing 150 nM hairpin DNA,

of phosphorylation process. The concentration of ammonium sul-fate and sodium hydrogen phosphate causing a 50% fluorescencedecrease was about 15 and 20 mM, respectively, which was com-parable with the literature values [4,20]. The salt effect on T4 PNKactivity could be tentatively explained based on the following rea-sons [46]. At high salt concentrations, the structure of hairpin DNAwas more stable, which probably inhibited the activity of the 5′-hydroxyl group. Also, the enzyme conformation might be affectedby a high concentration of salts, inducing the weakening of T4 PNKactivity or the affinity between T4 PNK and its substrates. Theseresults strongly indicated that the proposed strategy is promisingin quantitatively monitoring of the T4 PNK inhibitors.

4. Conclusions

In summary, a simple and sensitive strategy for monitoringT4 PNK activity and inhibition effects was developed by meansof a �-exo cleavage-induced DNAzyme releasing strategy. Thereleased DNAzyme unit from designed hairpin DNA probe after 5′-phosphorylation coupled with �-exo cleavage was used for cycliccleavage toward its MB substrate and offered an amplified sens-ing platform toward T4 PNK. The as-proposed method provideda relatively wide linear range from 0.005 to 0.2 U mL−1 and alow detection limit of 0.005 U mL−1 for T4 PNK activity analysis.Also, the inhibition effects of ADP, ammonium sulfate, and sodiumhydrogen phosphate on phosphorylation could be evaluated con-veniently. The developed method would be very useful for thecharacterization of polynucleotide kinases and their phosphoryla-tion reactions. It should be also easily extended for the detection ofmany other nucleic acid enzymes and may find widespread appli-cations in biological process researches, drug discovery, and clinicdiagnostics.

Acknowledgments

This work was funded by the National Natural Science Foun-dation of China (Nos. 21005043, 21175076 and 21375072), the

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cientific Special Expenditure for Non-profit Public Industry oftate Oceanic Administration (No. 201105020), and the Basicesearch Program of Qingdao (No. 13-1-4-214-jch), the Scienceoundation of China Postdoctor (No. 2012M511537).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.snb.2013.10.101.

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Biographies

Shufeng Liu received his Ph.D. degree in physical chemistry from the Institute ofChemistry, Chinese Academy of Sciences (China) in 2005. Now, he is an associateprofessor of chemistry in College of Chemistry and Molecular Engineering, QingdaoUniversity of Science and Technology, China. His research interests cover optical andelectrochemical biosensors.

Jingjing Ming is now a graduate student with research interest in fluorescentbiosensors in Qingdao University of Science and Technology, China.

Ying Lin is now a graduate student with research interest in electrochemical biosen-sors in Qingdao University of Science and Technology, China.

Chunfeng Wang is now a graduate student with research interest in electrochemicalbiosensors in Qingdao University of Science and Technology, China.

ctuato

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S. Liu et al. / Sensors and A

ao Liu is now a graduate student with research interest in optical biosensors iningdao University of Science and Technology, China.

huanbin Chen is now a graduate student with research interest in optical andlectrochemical biosensors in Qingdao University of Science and Technology, China.

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Feng Li received his Ph.D. degree in analytical chemistry from the Nankai Univer-sity (PR China) in 2005. Currently, he is a professor in the College of Chemistryand Pharmaceutical Sciences, Qingdao Agricultural University, China. His currentresearch interests cover analytical chemistry, bioelectrochemistry and chemicalbiosensors.