ORIGINAL PAPER

Detection of micrococcal nuclease for identifying Staphylococcusaureus based on DNA templated fluorescent copper nanoclusters

Taiping Qing1& Caicheng Long1

& Xuan Wang1& Kaiwu Zhang1

& Peng Zhang1& Bo Feng1

Received: 11 December 2018 /Accepted: 9 March 2019 /Published online: 18 March 2019# Springer-Verlag GmbH Austria, part of Springer Nature 2019

AbstractMicrococcal nuclease (MNase) is a naturally-secreted nucleic acid degrading enzyme with important role in thespread of the bacteria in an infected host. The content of MNase can be used to estimate the pathogenicity ofStaphylococcus aureus (S. aureus). A fluorometric method is described here for determination of the activity ofMNase and for identification of S. aureus using DNA templated fluorescent copper nanoclusters (CuNC). A double-stranded DNA (dsDNA) with AT-rich regions and protruding 3′-termini was identified as a high-selectivity substratefor MNase and as a template for CuNC. In the absence of MNase, the long AT-rich dsDNA templates the formationof CuNC that display bright yellow fluorescence, with excitation/emission peaks at 340/570 nm. However, thesubstrates are enzymatically digested to mononucleotides or short-oligonucleotide fragments, which fail to synthesizefluorescent CuNC. The method works in the 1.0 × 10−3 - 5.0 × 10−2 U mL−1 MNase activity range, has a1.0 mU mL−1 detection limit, and is highly selective over other exonucleases. The assay was successfully appliedto the detection of MNase secreted by S. aureus and to the identification of S. aureus.

Keywords Staphylococcus aureus . Nucleases . High-selectivity . Copper nanoclusters

Introduction

Staphylococcus aureus (S. aureus) is one of the major humanpathogens that can cause different kinds of illnesses [1–3]. Infact, S. aureus infection has become one of the most commoninfectious worldwide [4, 5]. In addition, S. aureus can alsosecrete a variety of toxins, which are responsible for mostcases of rapid-onset of food poisoning [6]. Therefore, rapidand accurate identification of S. aureus is critical for diagnosisand subsequent treatment of infectious diseases caused byS. aureus. The conventional methods for detecting S. aureus

mainly include culturing methods and metabolic tests [7–10].They can provide high sensitivity and precise results of detec-tion, but require longer microorganisms’ growth time and pro-fessional technical skills, which vastly limit their applicationsin emergency and critically ill situations. In order to overcomethe disadvantages of the culture-based techniques, some indi-rect protocols based on the amplification of specific nucleicacid sequences or recognition of toxins and other biomole-cules excreted by the pathogen are introduced [11–15].Among them, micrococcal nuclease (MNase) is a naturally-secreted nucleic acid degrading enzyme with important role inthe spread of the bacterial cells in the infected host [16, 17].MNase is a nonspecific endo-exonuclease that digests single-and double-stranded DNA and RNA, but it preferentially di-gests AT or AU-rich regions [18]. The content of MNase canbe used to evaluate the pathogenicity of S. aureus [19]. Due toits potential as a quick biomarker of S. aureus presence in thesample solutions, some MNase-based sensors for identifyingS. aureus have been reported [20–22]. For example, He et al.developed several simple fluorescent methods to detectMNase using electrostatic interaction-based fluorescence res-onance energy transfer (FRET) between quantum dots anddye-labeled single-stranded DNA (ssDNA) [23, 24]. Tang

Taiping Qing and Caicheng Long contributed equally to this work.

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s00604-019-3363-3) contains supplementarymaterial, which is available to authorized users.

* Taiping Qingtaiping_qing@163.com

* Bo Fengfengbo@xtu.edu.cn

1 College of Environment and Resources, Xiangtan University,Xiangtan 411105, Hunan Province, China

Microchimica Acta (2019) 186: 248https://doi.org/10.1007/s00604-019-3363-3

et al. reported an ultra-high sensitive strategy for MNase de-tection based onMNase-induced DNA strand scission and thedifference in affinity of grapheme oxide for ssDNA containingdifferent numbers of bases [25]. Although promising, thesetechniques suffer from intrinsic limitations such as the com-plicated synthesis procedure, the need of fluorophore-modified DNA substrates, and low energy transfer efficiencybetween the donor and the acceptor. In addition, most of thesubstrates in MNase-based sensors are ssDNA, which can bealso digested by Exo I or S1 nuclease and interfere the MNasedetection. Therefore, the development of simple and high-selectivity method for MNase analysis should be of generalinterest.

Recently, a novel fluorescent nanomaterial, DNA tem-plated copper nanocusters (DNA-CuNC), has been foundand studied in our group [26–28]. We have systematicallyinvestigated the effect of sequence composition on the forma-tion of fluorescent CuNC and found that double-strandedDNA templated CuNCwere poly(AT-TA)-dependence forma-tion [27]. Interestingly, the synthesis of DNA-CuNC is effi-cient and can be completed within several minutes under am-bient conditions. This facilitates the wide application of DNA-CuNC in biochemical analysis [29–32]. In addition, theMegaStokes shifting (230 nm) of DNA-CuNC (λex =340 nm, λem = 570 nm) maybe provided an opportunity fordetection of analyte from complex biological media [33–35].More importantly, the fluorescence intensity of DNA-CuNC is highly-dependent on the length of DNA,which holds an immense potential for nucleic acidlength-related enzymes.

Inspired by the challenge and significance of MNase detec-tion, we herein report a high selectivity strategy for evaluatingMNase activity and identifying S. aureus using DNA templatedCuNC. First, a smart DNA (dsDNA2), with AT-rich regions and3′ protruding termini, was designed for the high-selectivity sub-strates of MNase and template of CuNC. In the absence oftarget, the relatively long AT-rich dsDNA2 will induce the for-mation of CuNC with yellow fluorescence. However, the sub-strates undergo enzymatic digestion to mono or short-oligonucleotide fragments in the presence of MNase and thenfail to template fluorescent CuNC. Thus, theMNase activity canbe related to the changes in the fluorescence of the CuNC.

Experimental

Materials and reagents

All oligonucleotides used in this work were synthesized andHPLC-purified by Shanghai Sangon Biological EngineeringTechnology & Services Co. Ltd. (Shanghai, China. https://www.sangon.com/). The detailed sequences information ofis shown in Table S1. Exnuclease I (Exo I) and bovine

serum albumin (BSA) were purchased from ShanghaiSangon Biotechnology Co., Ltd. (Shanghai,China. https://www.sangon.com/). Micrococcal nuclease (MNase) and exo-nuclease III (Exo III) were purchased from TakaraBiotechnology Co., Ltd. (Dalian, China. http://www.takarabiomed.com.cn/). 3-(N-Morpholino) propanesulfonicacid (MOPS), sodium ascorbate (Vc), copper sulfate (Cu2+),and sodium chloride were commercially obtained fromDingguo Biotechnology Company, Ltd. (Beijing, China.http://dingguo.bioon.com.cn/), they are at least analyticalgrade and used without further treatment. MOPS buffer(10 mM MOPS, 150 mM NaCl, pH 7.8) was used for theformation of fluorescent CuNC. All stock and buffer wereprepared using autoclaved double-deionized water.

Apparatus

All fluorescence characterization of CuNC was performed onan F-7000 fluorescence spectrophotometer (Hitachi, Japan)with 0.2 × 1 cm2 quartz cuvette. Both the excitation and emis-sion slit were set at 5.0 nm, with a 700 V PMT voltage and a0.2 s response time. Fluorescence emission spectra of CuNCwere collected from 500 nm to 650 nm at room temperaturewith a 340 nm excitation wavelength. Fluorescence emissionphotographs of CuNC were obtained by a smartphone underUV irradiation. The morphology of the CuNC was character-ized by F20 transmission electron microscope with an accel-erating voltage of 200 kV. Samples were prepared by spincoating 10 μL of as-prepared CuNC onto carbon-coated cop-per grid substrates, which were then dried naturally overnight.

Detection of micrococcal nuclease (MNase) activity

All DNAwere first dissolved with ultrapure water to a com-mon final concentration of 10 μM, and then left at room tem-perature before use. In a typical procedure at the optimizedconditions, 1.0 μM probe DNA (P′) and 1.0 μM complemen-tary probe DNA (c-P′) was dissolved in MOPS buffer. Afterincubation for 10 min at room to ensure that nucleic acidswere hybridized to each other completely, different concentra-tion of MNase was added into the solution to give final vol-umes of 100 μL. Subsequently, the enzymatic cleavage reac-tion was carried out at 37 °C for 10 min. Finally, 3 μL sodiumascorbate of 100 mM, and 2 μL of copper sulfate of 10 mMwere introduced and triggered the formation reaction of fluo-rescent CuNC. The fluorescence spectra and emission imagesof the mixture were recorded at room temperature 5 min later.

Cleavage reaction by gel electrophoresis

In order to validate the enzymatic cleavage reaction ofMNase,the agarose gel electrophoresis assay was carried out. All thecontents in the sample were the same as that ofMNase activity

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detection but without the MOPS, sodium ascorbate, and cop-per sulfate. Then, different reaction solutions (10 μL) wereprestained with 2 μL SYBR Gold (100×). Subsequently, themixture solution was loaded on a 3% agarose gel for electro-phoresis. After running at 100 V for 30 min in a 0.5 × TBEbuffer (45 mM Tris-glacial acetic acid, 1 mM ethylene di-amine tetraacetic acid, pH 8.5) at room temperature, thephotographed of gel was obtained via a Bio-Red ImagerSystem.

Detection MNase in culture medium

Analysis of the target from complex samples is usually used totest the practical application capability of the proposed meth-od. As we wish to apply this nanoprobe to test the content ofMNase secreted by S. aureus, peptone culture medium is usedas the complex fluid and spiked with MNase at different con-centrations. Subsequently, the detection process of MNase inculture medium was the same as the steps in the buffer.

S. aureus culture and activity identification

The bacterial strains (S. aureus, CCTCCAB91093) andEscherichia coli (E. coli) were grown in a peptone culturemedium which contains 5 g L−1 yeast extract, 10 g L−1 pep-tone, and 5 g L−1 NaCl (pH 7.0). The culture medium wassterilized in high-pressure steam for 30 min at 120 °C beforeuse. Then the culture mediums containing S. aureus or E. coli,which were incubated for 0 h, 4 h, and 8 h, were centrifuged at5000 rpm for 5 min, respectively, and the supernatant wascollected and boiled for 10 min at 95 °C. This process candenature other protein and maintain the enzyme activity ofMNase. Supernatant (2 μL) was added into the nucleasedigestion reaction buffer (98 μL) which contains1.0 μM dsDNA2. Then, sodium ascorbate and coppersulfate were introduced and triggered the formation offluorescent CuNC. The solution was incubated for5 min at room temperature. Finally, the fluorescenceintensity of the incubated solution was measured at 570 nmwith excitation at 340 nm.

Results and discussions

Principle design

MNase is a thermostable nuclease and digests single- anddouble-stranded DNA or RNA, but preferentially hydrolyzeDNA or RNA occurs preferentially at AT or AU-rich regions[17, 36]. On the other hand, we have systematically investi-gated the effect of sequence composition on the formation offluorescent CuNC and found that both ssDNA and dsDNAcould template CuNC, but dsDNA templated CuNC were

poly(AT-TA)-dependence formation [26, 27]. Inspired by this,we aimed to design an AT-rich dsDNA as both the substratesof MNase and template of CuNC (dsDNA1), which can avoidthe interference of Exo I and template highlighter CuNC. Inorder to further avoid the interference of Exo III, which candigest blunt or recessed duplex DNAs [37], we extend a cou-ple of bases at two ends to form 3′ protruding terminus(dsDNA2). As shown in Scheme 1, when in the absence ofMNase, the relatively long AT-rich dsDNA2 will induce theformation of CuNC by addition of sodium ascorbate (Vc) andcopper sulfate (Cu2+). Similarly, the dsDNA2 can also synthe-size CuNC in the presence of other exonuclease results in highfluorescence intensity. In contrast, MNase can hydrolyzethe AT-rich dsDNA2 and consequently produce monoor short-oligonucleotide fragments, which fail to synthe-size fluorescent CuNC. The concentration of MNase canbe quantitatively monitored according to the change offluorescence signal.

Screening of the high-selectivity substrates

Inspired by the challenge and significance, we aimed to designand screen a high-selectivity substrate for MNase. First of all,three different type of DNA (ssDNA1, dsDNA1, dsDNA2)are designed and used as both the substrates of MNase andtemplate of CuNC. The sequences of all DNAs used in thiswork are listed in Table S1. The fluorescence intensity of theCuNC formed is used to determine the responses of differentDNAs to different exonuclease. As shown in Fig. 1, ssDNA1can be digested by Exo I and MNase, resulting in low selec-tivity in MNase assays. Complete complementary dsDNA(dsDNA1) can also be excised in the presence of Exo III orMNase, lacking absolute selectivity in MNase detection.Attractively, 3′ protruding terminus dsDNA (dsDNA2) willbe only digested by MNase, with negligible interference fromother exonuclease. Those results show that dsDNA2 hashigh specificity towards MNase digestion and is a high-efficiency template for CuNC formation. Thus, dsDNA2is screened as the high-selectivity substrate of MNase inMNase detection.

Feasibility verification of the method

Following the design, the feasibility of the proposed methodfor MNase assay was investigated. First, the dsDNA tem-plated CuNC were confirmed by fluorescence spectrum andtransmission electron microscopy (Fig. S1). Then, the detec-tion of MNase of S. aureus was characterized by CuNC’ fluo-rescence. As shown in Fig. 2a, the solution in the absence ofMNase produce obvious fluorescence in the spectra region ofabout 570 nm (curve a), while the fluorescence of the solutioncontaining MNase is negligible (curve b). In order to testifywhether the MNase protein itself quench the fluorescence of

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CuNC, the denatured MNase (high-pressure steam for 30 minat 120 °C) was instead of the active MNase. The solutiontreated with denatured MNase exhibit high fluorescence(curve c), which is slightly lower than that dsDNA′ presentwithout any treatment (curve a). Fluorescence emission pho-tographs of formed CuNC under UV lamp excitation are ac-cordance with the fluorescent results. In addition, agarose gelelectrophoresis analysis of the products was also investigatedto verify the feasibility. As shown in Fig. 2b, when dsDNA2was not undergone any treatment, a significant electrophoresisband can be identified (lane a). However, in the presence ofMNase, the dsDNA2 was digested completely by MNaseand no electrophoresis bands will be visualized (lane b).When the dsDNA2 was treated with denatured MNase,the same mobility is observed (lane c). These resultsindicate that the DNA-CuNC based strategy for MNasedetection is also feasible.

Optimization of experimental conditions

In order to achieve a better detection performance, the substrate(dsDNA2) concentration and hydrolysis time were optimized bythe ratio of signal change ((F0− F)/F0), where F0 is the fluores-cence intensity of the detection system in the absence of MNaseand F is that in the presence of MNase. As shown in Fig. S2a,with the increase of dsDNA2 concentration, the fluorescence in-tensity of sensing system increase at first and then increase slowlyafter 1μM. Fig. S2b shows that the hydrolysis process is very fastand reaches maximum saturation in 10 min, which potentiallyindicate that the proposed method is fast to the MNase. So,1 μM of dsDNA2 and 10 min hydrolysis time are chosen forsubsequent experiment. Besides, the effects of Vc concentrationand Cu2+ concentration on CuNC’ formation was also investigat-ed. As a result, 3 mM Vc and 300 μM Cu2+ are chosen as theoptimal condition for the detection system (Fig. S3).

Scheme 1 Schematic illustrationof micrococcal nuclease (MNase)assay based on smart DNAtemplated CuNC

Fig. 1 aGeneral types of nucleic acid substrate and their response for different exonuclease; b normalized fluorescence intensity of different DNAs aftertreating with different exonuclease, the concentration of ssDNA1, dsDNA1 and dsDNA2 were all 1.0 μM

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MNase detection in buffer with smart DNA-CuNC

After the feasibility verification and optimization of condi-tions, the analytical performance of the proposed strategy forMNase detection is evaluated. The dynamic response rangeand the detection capability are indentified by CuNC’ fluores-cence. As shown in Fig. 3a, with the increase of MNase con-centration, the fluorescence intensity of detection system de-creases gradually, indicating a gradual cleavage of dsDNA2.A linear dependence (R2 = 0.9807) between the F/F0 andMNase concentration is acquired in the range from 1.0 ×10−3 - 5.0 × 10−2 U mL−1, with the detection limit of1.0 mU mL−1 (Fig. 3b). The linear equation will be expressedas F/F0 = 0.9581–11.552x, where F/F0 is the ratio of fluores-cence intensity of CuNC at 570 nm in the presence and ab-sence of MNase. This detection capability is comparable to oreven better than reported methods (Table S2) [20–24], indi-cating this smart DNA-CuNC based method is a promisingmethod towards MNase detection.

Selectivity study

As mentioned above, available highly selective fluorescentprobes for MNase are very limited. Most of the nucleic acidsubstrates in MNase-based sensors are ssDNA, which can bealso digested by Exo I or S1 exonuclease and interfere theMNase detection. To gauge the recognition specificity of thisnanoprobe to MNase, the fluorescence responses of the DNA-CuNC to interferent substances such as BSA, Exo I, and Exo IIIwere investigated. As illustrated in Fig. 4, the fluorescenceintensity of the sensing system nearly remain unchanged inthe presence of various interferent substances as compared tothe blank sample. The direct-viewing effect of selectivity studywas further confirmed by optical imaging. From fluorescenceemission images, the fluorescence is quenched in the presenceof MNase, while there is light yellow-emission fluorescence inthe presence of various interferent substances (inset Fig. 4b).These results clearly show that smart DNA-CuNC-based strat-egy is highly selective for MNase over other exonuclease.

Fig. 3 a Fluorescence spectra of the detection system with various concentrations of MNase. b Relationship of F/F0 with MNase concentration, Ex/Em= 340/570 nm, Inset shows the linear response of the detection system to MNase

Fig. 2 a Fluorescence spectra ofthis sensing system for MNaseunder different reactionconditions: (a) dsDNA2 +Vc +Cu2+; (b): dsDNA2 +MNase +Vc +Cu2+; (c) dsDNA2 + dena-tured MNase + Vc +Cu2+; Inset:the corresponding fluorescenceemission photograph under UVlamp excitation. b Agarose gelelectrophoresis analysis of thedifferent products as (a). The con-centration of dsDNA2, MNase,Vc and Cu2+ were 1 μM, 0.001 U/μL, 3 mM and 300 μM,respectively

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Detection of MNase in complex sample

Since the MegaStokes shifting of DNA-CuNC was 230 nm(Ex/Em= 340/570 nm), it maybe provide an opportunity fordetection of the target from complex biological media. Toevaluate the practical application capability of this strategy,the MNase-spiked culture medium is used to investigate thefeasibility for nuclease detection from complex biologicalfluids. As shown in Fig. 5, the fluorescence intensity ofCuNC also decreases gradually with increasing MNase con-centration in dilute culture medium. This result exhibits sim-ilar trend to that in reaction buffer, suggesting that the methodis reliable and practical for the assay of the target fromreal complex sample. On the other hand, the UV lightused for fluorescence excitation will be screened off byUV absorbers and this will weaken the signal, the de-velopment and application of near-infrared fluorescentnanoclusters is also currently being under taken in ourlaboratory.

Detection of MNase secreted by S. aureus

It was reported that the content of MNase can be used toevaluate the pathogenicity of S. aureus. So, the detection ofMNase secreted by S. aureuswas also investigated. Firstly, theculture mediums containing S. aureus or E. coli were collect-ed. Then, the fluorescence intensity of the CuNC-basednanoprobe in different culture mediums was recorded. Asshown in Fig. 6, the fluorescence intensity of the DNA-CuNC is gradually increased with the increase of the incubat-ed time (with the increase of MNase) in S. aureus containedculture mediums, but not a significant change in E. colicontained culture mediums. This result shows that thisnanoprobe can be used to detect MNase in practical samplessensitively, and might be a promising probe for identifyingS. aureus. Although promising, our approach still needs ashort bacterial culture to identify S. aureus. The eliminationor minimal need for bacterial cultures remains an attractivechallenge for reducing detection times.

Fig. 4 Selectivity of the detection system for MNase assays; aFluorescence emission spectra of the detection system with and withoutMNase and interferent substances; b Fluorescence intensity of CuNC in

the presence of MNase and various interferent substances; inset: thecorresponding fluorescence emission photograph under UV lamp

Fig. 6 Comparison of MNase activity in different cell-free extracts, fluo-rescence intensity of CuNC-based nanoprobe in different culture me-diums containing S. aureus or E. coli (incubated for 0 h, 4 h, and 8 h)

Fig. 5 Fluorescence emission spectra of smart DNA-CuNC in culturemedium in the presence of increasing MNase concentration, the inset isthe corresponding fluorescence emission image under UV lamp

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Conclusions

A highly selectivity fluorometric method for MNase detectionis developed based on DNA templated formation of coppernanoclusters (CuNCs). After rational design, a smart dsDNA,with AT-rich regions and 3′ protruding termini (dsDNA2), isscreened as the high-selectivity substrates of MNase and tem-plate of CuNC. In the prescence of MNase, the long AT-richdsDNA2 is cleaved into short oligonucleotide fragments,which fail to synthesize fluorescent CuNC. The MNase activ-ity can be related to the changes in the fluorescence of theCuNC. The proposed method exhibits low detection limitand high selectivity for MNase over other exonucleases.Thanks to its excellent analytical performance, this detectionsystem is successfully applied in the detection of MNase inculture medium. Furthermore, the proposed method is alsoextended to detecting MNase secreted by S. aureus and iden-tifying S. aureus. Therefore, we believe that this proposedstrategy may pave a new way to develop simple and highselectivity methods for environmental and clinicalapplications.

Acknowledgements This work was supported by the National NaturalScience Foundation of China (21777135 and 51708475), the NaturalScience Foundation of Hunan Province, China (2019JJ50596,2019JJ40283, and 2018JJ3496), and Hunan 2011 CollaborativeInnovation Center of Chemical Engineering & Technology withEnvironmental Benignity and Effective Resource Utilization.

Compliance with ethical standards The author(s) declarethat they have no competing interests.

References

1. Jarraud S, Mougel C, Thioulouse J, Lina G, Meugnier H, Forey F,Nesme X, Etienne J, Vandenesch F (2002) Relationships betweenStaphylococcus aureus genetic background, virulence factors, agrgroups (alleles), and human disease. Infect Immun 70:631–641

2. Daum RS (2007) Skin and soft-tissue infections caused bymethicillin-resistant Staphylococcus aureus. N Engl J Med 357:380–390

3. Suaifan GA, Alhogail S, Zourob M (2017) Rapid and low-costbiosensor for the detection of Staphylococcus aureus. BiosensBioelectron 90:230–237

4. TörökME, Day NP (2014) Staphylococcal and streptococcal infec-tions. Medicine 42:1–7

5. Venkataramana M, Kurkuri MD (2016) Simple quantum dotbioprobe/label for sensitive detection of Staphylococcus aureusTNase. Sensor Actuators B Chem 222:1201–1208

6. Otto M (2014) Staphylococcus aureus toxins. Clin Microbiol 17:32–37

7. Böcher S, Smyth R, Kahlmeter G, Kerremans J, Vos MC, Skov R(2008) Evaluation of four selective agars and two enrichment brothsin screening for methicillin-resistant Staphylococcus aureus. J ClinMicrobiol 46:3136–3138

8. Wang J, Wu X, Wang C, Shao N, Dong P, Xiao R, Wang S (2015)Magnetically assisted surface-enhanced Raman spectroscopy forthe detection of Staphylococcus aureus based on aptamer recogni-tion. ACS Appl Mater Interfaces 7:20919–20929

9. Xiong J,WangW, Fu Z (2017) Fluorimetric sandwich affinity assayfor Staphylococcus aureus based on dual-peptide recognition onmagnetic nanoparticles. Microchim Acta 184:4197–4202

10. Jang H, Lee P, Kim S, Kim SM, Jeon TJ (2017) An antibacterialmicrofluidic system with fish gill structure for the detection ofStaphylococcus via enzymatic reaction on a chromaticpolydiacetylene material caused by lysostaphin. Microchim Acta184:4563–4569

11. Shi J, Chan C, Pang Y, Ye W, Tian F, Lyu J, Zhang Y, Yang M(2015) A fluorescence resonance energy transfer (FRET) biosensorbased on graphene quantum dots (GQDs) and gold nanoparticles(AuNPs) for the detection of mecA gene sequence ofStaphylococcus aureus. Biosens Bioelectron 67:595–600

12. Hernandez FJ, Huang L, Olson ME, Powers KM, Hernandez LI,Meyerholz DK, Thedens DR, Behlke MA, Horswill AR,McNamara JO II (2014) Noninvasive imaging of Staphylococcusaureus infections with a nuclease-activated probe. Nat Med 20:301–306

13. Flenker KS, Burghardt EL, Dutta N, BurnsWJ, Grover JM, KenkelEJ, Weaver TM, Mills J, Kim H, Huang L, Owczarzy R,Musselman CA, Behlke MA, Ford B, McNamara JO II (2017)Rapid detection of urinary tract infections via bacterial nucleaseactivity. Mol Ther 25:1353–1362

14. Wang J, Li H, Li T, Ling L (2018) Determination of bacterial DNAbased on catalytic oxidation of cysteine by G-quadruplexDNAzyme generated from asymmetric PCR: application to the col-orimetric detection of Staphylococcus aureus. MicrochimActa 185:410–418

15. Ning Y, Zou L, Gao Q, Hu J, Lu F (2018) Graphene oxide-based fluorometric determination of methicillin-resistantStaphylococcus aureus by using target-triggered chain reac-tion and deoxyribonuclease-assisted recycling. MicrochimActa 185:183–190

16. Heins JN, Suriano JR, Taniuchi H, Anfinsen CB (1967)Characterization of a nuclease produced by Staphylococcus aureus.J Biol Chem 242:1016–1020

17. Olson ME, Nygaard TK, Ackermann L, Watkins RL, Zurek OW,Pallister KB, Griffith S, Kiedrowski MR, Flack CE, Kavanaugh JS,Kreiswirth BN, Horswill AR, Voyich JM (2013) Staphylococcusaureus nuclease is a SaeR/S-dependent virulence factor. Infectimmun IAI-01242

18. HörzW, AltenburgerW (1981) Sequence specific cleavage of DNAby micrococcal nuclease. Nucleic Acids Res 9:2643–2658

19. Lagacé-Wiens PR, Alfa MJ, Manickam K, Karlowsky JA (2007)Thermostable DNase is superior to tube coagulase for direct detec-tion of Staphylococcus aureus in positive blood cultures. J ClinMicrobiol 45:3478–3479

20. Sato S, Takenaka S (2014) Highly sensitive nuclease assays basedon chemically modified DNA or RNA. Sensors 14:12437–12450

21. He Y, Jiao B (2015) Simple and convenient G-quadruplex-basedfluorescence assay of micrococcal nuclease activity. RSC Adv 5:104116–104121

22. Li D, Qin J, Yan G (2018) A phosphorescent sensor for detection ofMicrococcal nuclease base on phosphorescent resonance energytransfer between quantum dots and DNA-ROX. Sens Actuators BChem 255:529–535

23. Qiu T, Zhao D, Zhou G, Liang Y, He Z, Liu Z, Peng X, Zhou L(2010) A positively charged QDs-based FRET probe for micrococ-cal nuclease detection. Analyst 135:2394–2399

24. Huang S, Xiao Q, He ZK, Liu Y, Tinnefeld P, Su XR, Peng XN(2008) A high sensitive and specific QDs FRET bioprobe forMNase. Chem Commun (45):5990–5992

Microchim Acta (2019) 186: 248 Page 7 of 8 248

25. He Y, Xiong LH, Xing XJ, Tang HW, Pang DW (2013) An ultra-high sensitive platform for fluorescence detection of micrococcalnuclease based on grapheneoxide. Biosens Bioelectron 42:467–473

26. Qing Z, He X, He D, Wang K, Xu F, Qing T, Yang X (2013) Poly(thymine)-templated selective formation of fluorescent coppernanoparticles. Angew Chem Int Ed 52:9719–9722

27. Qing T, Qing Z, Mao Z, He X, Xu F, Wen L, He D, Shi H, Wang K(2014) dsDNA-templated fluorescent copper nanoparticles: poly(AT-TA)-dependent formation. RSC Adv 4:61092–61095

28. Qing Z, Qing T, Mao Z, He X, Wang K, Zou Z, Shi H, He D (2014)dsDNA-specific fluorescent copper nanoparticles as a Bgreen^nano-dye for polymerization-mediated biochemical analysis.Chem Commun 50:12746–12748

29. Qing Z, Zhu L, Yang S, Cao Z, He X, Wang K, Yang R (2016) Insitu formation of fluorescent copper nanoparticles for ultrafast zero-background Cu2+ detection and its toxicides screening. BiosensBioelectron 78:471–476

30. Liu R, Wang C, Hu J, Su Y, Lv Y (2018) DNA-templated coppernanoparticles: versatile platform for label-free bioassays. TRACTrend Anal Chem 105:436–452

31. Ge J, Dong ZZ, Bai DM, Zhang L, Hu YL, Ji DY, Li ZH (2017) Anovel label-free fluorescent molecular beacon for the detection of3′-5′ exonuclease enzymatic activity using DNA-templated coppernanoclusters. New J Chem 41:9718–9723

32. Dong ZZ, Zhang L, Qiao M, Ge J, Liu AL, Li ZH (2016) A label-free assay for T4 polynucleotide kinase/phosphatase activity and its

inhibitors based on poly (thymine)-templated copper nanoparticles.Talanta 146:253–258

33. Zhang X, Liu Q, Jin Y, Li B (2019) Determination of the activity ofT4 polynucleotide kinase phosphatase by exploiting the sequence-dependent fluorescence of DNA-templated copper nanoclusters.Microchim Acta 186:3–11

34. Zhang Y, Chen Z, Tao Y, Wang Z, Ren J, Qu X (2015)Hybridization chain reaction engineered dsDNA for Cu metalliza-tion: an enzyme-free platform for amplified detection of cancercells and microRNAs. Chem Commun 51:11496–11499

35. Zhang L, Cai QY, Li J, Ge J, Wang JY, Dong ZZ, Li ZH (2015) Alabel-free method for detecting biothiols based on poly (thymine)-templated copper nanoparticles. Biosens Bioelectron 69:77–82

36. Martensen TM, Stadtman ER (1982) Micrococcal nuclease cleav-age of nucleotide linked to glutamine synthetase yieldsphosphotyrosine at the ligation site. P Nat Acad Sci USA 79:6458–6460

37. Qing T, He D, He X, Wang K, Xu F, Wen L, Shangguan J, Mao Z,Lei Y (2016) Nucleic acid tool enzymes-aided signal amplificationstrategy for biochemical analysis: status and challenges. AnalBioanal Chem 408:2793–2811

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