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Biosensors and Bioelectronics 26 (2011) 3488–3493

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

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ptical analysis of lactate dehydrogenase and glucose by CdTe quantum dots andheir dual simultaneous detection

iuqing Yanga,b, Xiangling Rena, Xianwei Menga, Hongbo Lia,b, Fangqiong Tanga,∗

Laboratory of Controllable Preparation and Application of Nanomaterials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, and Graduate University of thehinese Academy of Sciences, Beijing 100190, People’s Republic of ChinaGraduate School of the Chinese Academy of Sciences, Beijing 100049, People’s Republic of China

r t i c l e i n f o

rticle history:eceived 23 November 2010eceived in revised form 24 January 2011ccepted 24 January 2011vailable online 2 February 2011

eywords:uantum dots

a b s t r a c t

Biomolecules detection by size-controlled quantum dots (QDs) was promising in developing clinic diag-nostic techniques. In this work, a novel bioanalytical platform was developed to detect the activity ofnicotinamide adenine dinucleotide (NAD) dependent enzyme, lactate dehydrogenase (LDH), and the con-centration of glucose by the changes of fluorescence intensities of the QDs based on the electron transferbetween QDs and sensitive biomolecules. The fluorescence intensities of the QDs was firstly quenchedby NAD and then intensified with increasing amounts of the LDH because of the consumption of the NADby the biocatalyzed reaction. Also the glucose led to the decline of fluorescence due to the formation

actate dehydrogenaselucoseluorescenceual simultaneous detection

of hydrogen peroxide (H2O2) which was the product of the glucose reacting with the glucose oxidase(GOD). The linear calibration plots of the activity of LDH and glucose were obtained from 250 to 6000 U/Land 1.67 to 6.67 mM, respectively. The detection system was also successfully applied to detect LDH andglucose in human serum samples. This analysis process was very convenient and simple because the QDsneed not to be modified by any organic or biological molecules before they were used into the system.Moreover, the established method had great potential in detection of the physiological level of some

iagno

biomolecules in clinical d

. Introduction

Quantum dots (QDs) have been widely used in immunoas-ays, molecular imaging, and in vivo biological labels due to theirnique photophysical properties, such as broad absorption, nar-ow emission and high quantum yield (Medintz et al., 2005; Lit al., 2007). Recently, an increasingly growing field of researchs focused on developing optical sensors based on the change ofuorescent intensities (Algar and Krull, 2008; Yuan et al., 2009).ome QDs-based biosensors have been developed, including thoseor DNA hybridizations (Goldman et al., 2002; Kim et al., 2007; Cuit al., 2008; Marin and Merkoci, 2009), proteins and antigen (Gaot al., 2004; Choi et al., 2006; Ao et al., 2006), cancerous cells (Cait al., 2006; Hu et al., 2006; Yong, 2009) and virus (Deng et al.,007; Zhang et al., 2007). Whereas, the growing need for multi-lexing and miniaturization of biological/chemical analysis has led

o major research efforts to develop novel detection technologies,hich have enabled simultaneous screening of multiple samplesith high throughput (He et al., 2010). To the best of our knowledge,uman health is always indicated by various physiologically impor-

∗ Corresponding author. Tel.: +86 10 82543521; fax: +86 10 62554670.E-mail address: tangfq@mail.ipc.ac.cn (F. Tang).

956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2011.01.031

stics of various diseases.© 2011 Elsevier B.V. All rights reserved.

tant species together, such as glucose, uric acid, cholesterol andsome enzymes. So disease diagnosis on the basis of biomolecularanalysis requires sensitive, cost-effective, and multiplexed assays.The unique size-dependent properties of QDs make this materialcould offer a new powerful tool for biosensing and biorecognitionevents in many areas of analysis (Klimov et al., 2000; Sun et al.,2007; Smith and Nie, 2009; Zrazhevskiya and Gao, 2009). The sizecontrolled QDs have been used to achieve the multiplex analysis ofprotein residues (Kim et al., 2009), toxin (Goldman et al., 2004) andmetal ions, such as Hg+ and Ag+ by nucleic acid modified CdSe/ZnS(Freeman et al., 2009a,b). However, the application of QDs for multi-plex determination of physiological substrate, for example, enzymeactivities and some important species synchronously is still scarce.

Herein we choose the lactate dehydrogenase (LDH) and glucoseto fabricate a bioanalytical platform which can offer advantagesin terms of sensitivity, less analysis time, dual simultaneous detec-tion for analyzing important physiological species. LDH and glucoseare two of the most common physiological indexes of biochemi-cal analysis in disease diagnosis and they represent two different

kinds of enzyme reactions in metabolism. LDH, as one of the nicoti-namide adenine dinucleotide (NAD) dependent enzyme, is one ofthe most physiologically important species in clinical diagnosis ofvarious diseases, such as congestive heart diseases, hepatitis aswell as liver cancer (Kato et al., 2006). Our previous research has

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evealed that the activity of LDH could be detected by the QDs-ased biosensor (Ren et al., 2010). The level of glucose also plays an

mportant role in reflecting the metabolic disorder. The ability tobtain rapid, accurate and precise glucose measurement is essentialo the appropriate administration of insulin, and diabetes therapyLuxton, 1993; Ren et al., 2005a,b). Thus these two representativepecies detected by the size-controlled fluorescence features of QDsould offer a new method for carrying out analytical and diagnosticrocedures.

In this work, our researches mainly focus on a strategy to ana-yze the activity of LDH and glucose by QDs respectively. The newetection system is also used for the determination of LDH andlucose in human serum. Furthermore, a bioanalytical platform issed to detect LDH and glucose synchronously by using two dif-erent fluorescence features of CdTe QDs. Compared with previouspplications of QDs to prepare optical biosensor, our method hast least three main advantages over the existing methods: (i) ThedTe QDs are synthesized in aqueous solution and then directlymployed as optical probe without any modification. The analyti-al process turns out to be convenient and simple. (ii) The CdTe QDsan be used in several enzyme detection systems. This will greatlyimplify the assembling process of the detection. (iii) The novel bio-nalytical platform has a possibility of multiplexed analysis by theize-tunable fluorescence emission. Furthermore, our protocol pro-ides a generic way to analyze numerous NAD-dependent enzymesnd H2O2-generative substrates among physiological species syn-hronously.

. Experimental

.1. Materials

All reagents were of analytical grade and used without furtherurification. Lactate dehydrogenase (LDH, 30 U/mg) and nicoti-amide adenine dinucleotide (NAD) were purchased from Shanghaiayon Biological Technology Company and l-lactate (LL) was fromlfa Aesar Company. Glucose oxidase (GOD) was extracted fromspergillus niger (Sigma company, 118 U/mg). �-d-Glucose wasurchased from Sigma. Phosphate buffer containing 0.1 M KCl con-isted of KH2PO4 and Na2HPO4 (0.2 M, pH = 7.0).

.2. Synthesis of CdTe QDs

CdTe QDs were obtained via a method described in the liter-ture with minor modification (Gaponik et al., 2002). Briefly, thedTe precursor solution was prepared by adding freshly preparedaHTe solution to a nitrogen-saturated CdCl2 solution at pH 11.5

n the presence of thiolglycolic acid (TGA) as stabilizer. The precur-or concentrations were [Cd] = 10 mM, [TGA] = 14 mM, [Te] = 5 mMespectively. The CdTe precursor solution was heated at 90 ◦C forarious times. A series of QDs with sizes ranging from 2 to 4 nmere obtained. In this study, CdTe QDs with different emission peakosition at 545 nm, 570 nm and 600 nm respectively were used.

As shown in Fig. S1(A) (see the Supporting information), thebvious UV–vis absorption peaks (curve a, b) which indicate thathe CdTe QDs are closed to monodispersed. The fluorescence emis-ion maximum appears at 570 nm and 600 nm respectively. Thehotoluminescence full widths at half-maximum are about 43 nmnd 47 nm for QD570 (Emission wavelength is 570 nm), QD600Emission wavelength is 600 nm) respectively. The concentrations

f the QD570 and QD600 are 0.58 × 10−6 M and 1.48 × 10−6 Mespectively (Yu et al., 2003). The X-ray Diffraction (XRD) patternsf the CdTe QDs are shown in Fig. S1(B) (see the Supporting infor-ation). The peak in the (1 1 1) direction is at 24.4◦. The XRD results

ndicate that QDs have fine nanocrystal structure which is con-

tronics 26 (2011) 3488–3493 3489

sistent with the reported results (Lee et al., 2008). As shown inFig. S1(C) (see the Supporting information), Transmission electronmicroscope (TEM) images also suggest that the CdTe QDs have anarrow size distribution. The average size of the CdTe QDs is 3.0 nm.

2.3. Instrumentation

Transmission electron microscope (TEM) of QDs nanoparticleswas obtained with a JEM-2100 electron microscope, operating at160 kV. The photoluminescence spectra were recorded by a CaryEclipse fluorescence spectrophotometer (Varian, Inc.). If not spe-cially stated, the samples were excited at 450 nm, and the excitingslit and the emission slit were 5 and 5 nm, respectively. The opti-cal properties of solutions were measured using quartz cuvettesof 10 mm path length and a standard solid sample holder, respec-tively.

2.4. Fluorescence experiments

The detection procedure was described as follows: 400 �L ofQDs (0.4 × 10−5 M) was dissolved into 2 mL by PBS buffer (pH = 7.0)and then reacted with different concentrations of NAD solution (theconcentration is from 0 to 8 mM) for 5 min, then the fluorescenceintensities of the solutions were recorded. The detection procedureof the activity of LDH was the same as the previous description.After adding NAD into the QDs solution, then the equal molaramount of substrate LL and the different amounts of enzymes werealso added, and the mixture was reacted for 10 min. The detectionof glucose was the same as the above procedure. 400 �L of QDswas dissolved into 2 mL by PBS buffer (pH = 7.0) with 2 U GOD anddifferent concentrations of glucose solution (the concentration wasfrom 1.11 to 8.33 mM) for 10 min. Then the fluorescence intensitiesof the solution were recorded by the fluorescence spectrophotome-ter, and the wavelength � = 450 nm of the laser source was used forthe excitation of the QDs detection system. The fluorescence signalwas recorded over a range from � = 480 nm to � = 630 nm.

2.5. Dual detection system for LDH and glucose

For the chip-based analyses, a multi-chamber microcup(3 mm × 3 mm) was prepared on the glass slide by photo-masklithography (Hiraoka and Welsh, 1984) (see from Supportinginformation Fig. S2). Two PBS buffer (pH = 7.0) solutions (5 �L) con-taining two different kinds of QDs detection system were droppedinto two chambers of the microcup, respectively. The analytecontaining both LDH and glucose were added into the two cham-bers. Finally, the fluorescence profiles of the two chambers wereobtained simultaneously from the fluorescence spectrophotometerand the color images were recorded by camera.

3. Results and discussion

3.1. Detection of the activity of LDH

Fig. 1A shows typical fluorescence intensities curves obtainedfor CdTe QD570 in the present of different activity units (U/L) ofLDH at optimal condition. Each sample is analyzed by reacting QDswith different amounts of LDH in the present of coenzyme NADand the substrate l-lactate (LL) for a fixed time interval of 10 min.It can be seen that the fluorescence is intensified by the increas-ing amounts of LDH. The inner plot depicts the calibration curve

that corresponds to the fluorescence intensities ratio F/F0 (F and F0refer to the fluorescence intensities of QDs in the presence/absenceof analyst) of QD570 upon the different amounts of LDH. It revealsdetectable concentration range of 250–6000 U/L and the correla-tion coefficient is 0.996. To the best of our knowledge, the normal

3490 L. Yang et al. / Biosensors and Bioelectronics 26 (2011) 3488–3493

Fig. 1. (A) Fluorescence changes upon the interaction of CdTe QD570 with differentamounts of LDH: (a) before addition of the LDH; (b)–(g) the amount of the LDH is 0.5,1, 3, 6, 12, 24 U in 2 ml solution respectively). The inner plot is the linear curve corre-sponds to the optical analysis of different amounts of LDH by the CdTe/CdS QDs. Eachsample with 1 mM NAD and 1 mM LL is analyzed after the CdTe QDs reacting withvariable amounts of LDH for 10 min. (B) Fluorescence changes upon the interactiono(cN

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f CdTe QD570 with different concentrations of NAD: (a) before addition of the NAD;b)–(e) the concentration of the NAD is 1, 2, 4, 8 mM respectively. The inner is thealibration curve corresponds to the optical analysis of different concentrations ofAD by the CdTe QDs.

mount of the LDH in human blood serum is 100–300 U/L, Whenhe amount of LDH is >1000 U/L, it may be some differentiating dis-rders. So we can qualitatively detect the amounts of LDH by thehanges of the fluorescence intensities of the QDs.

In order to prove the principle of optical analysis of LDH, thehanges of fluorescence intensities quenched by different concen-rations of NAD are also measured. It has been reported that NADould quench the fluorescence of the QDs by an electron transferET) process (Freeman and Willner, 2009). From Fig. 1B, we can seehat the fluorescence intensities of the QD570 turn to decline withhe increment of the concentration of NAD. The inner plot shows thealibration curve that corresponds to the changes of fluorescencentensities ratio F/F0 of the QD570 upon treatment with differentoncentrations of NAD.

.2. The principle of LDH detection

In the biochemical reaction, the LDH catalyzes the intercon-ersion of l-lactate and pyruvate with the coenzyme NAD. Theiochemical reaction of the LDH occurs as the following:

-lactate + NAD+ LDH−→Pyruvate + NADH + H+ (1)

ig. 2 illustrates the principle of LDH detection by QDs. NAD, and itseduced form, nicotinamide adenine dinucleotide (NADH), knowns coenzymes, the two nucleotides transfer hydrogen atoms and

Fig. 2. Schematic principle for assay of NAD (A) and the activity of the LDH (B) basedon the electron transfer of QDs and the biochemical reaction.

electrons from one metabolite to another in many cellular redoxreactions (Xie et al., 2009). In our system, the NAD originallydiffuses to the surface of CdTe QDs by chemical adsorption or elec-trostatic force and affects the surface chemical bond of the QDs.The fluorescence of the QDs is quenched by the NAD through elec-tron transfer (ET) process (Fig. 2(A)). Control experiments provedthat both the substrate LL and the NADH, the reduced form of NAD,do not affect the fluorescence intensities of QDs, respectively (seeSupporting information Fig. S3). So the proposed biochemical reac-tion during the LDH detection is shown in Fig. 2(B). When theenzyme LDH and the substrates LL are added into the detectingsystem, the biochemical reaction might consume more NAD. As aresult, fewer NAD are used to interact with the QDs and the fluo-rescence intensities of QDs do not decline remarkably. The moreamount of LDH, the more consumed NAD, so the fluorescence ofthe QDs is intensified with increasing amount of the LDH.

3.3. Detection of concentration of glucose

Besides the enzyme, CdTe QDs is also applied to detect a com-mon physiological substrate, glucose. Fig. 3A exhibits the variousfluorescence spectra of CdTe QD600 for different concentrations ofglucose. The fluorescence spectra are taken after the QD600 react-ing with GOD and variable concentrations of glucose for 10 min inphosphate buffer. As the concentration of glucose increases grad-ually, the quenching of the fluorescence intensities of CdTe QD600are observed. The inner plot depicts the calibration curve that cor-responds to the fluorescence intensities ratio F/F0 (F and F0 refer tothe fluorescence intensities of QDs in the presence/absence of ana-lyst) of QDs upon the different concentrations of glucose. The linearplot for glucose concentration range from 1.67 to 6.67 mM is deter-mined with the correlation coefficient 0.992. The result reveals thatthe sensitive QDs based sensor can work well in glucose solution.

In order to prove the principle of optical analysis of glucose,the changes of fluorescence intensities quenched by different con-centrations of hydrogen peroxide (H2O2) are also measured. Manyreports have shown that QDs are sensitive to H2O2 which is theproduct of the glucose reacting with the enzyme GOD (Gill et al.,2008; Cao et al., 2008). The result from the Fig. 3B shows that thefluorescence intensity of QDs is quenched by the increasing con-centration of H2O2. The inner plot shows the calibration curve thatcorresponds to the changes of fluorescence intensities of the QD600upon treatment with different concentrations of H2O2.

3.4. The principle of glucose detection

Based on the previous researches, the schematic sensing of glu-cose detection by QDs is shown in Fig. 4.

L. Yang et al. / Biosensors and Bioelec

Fig. 3. (A) Fluorescence changes upon the interaction of CdTe QD600 with differ-ent concentrations of glucose: (a)–(i) the concentration of the glucose is 1.11, 1.67,2.22, 2.78, 3.33, 4.44, 5.55, 6.67, 8.33 mM respectively. The inner plot is the linearcurve corresponds to the optical analysis of different concentrations of glucose bythe CdTe QDs. Each sample is analyzed after the CdTe QDs with GOD and variableconcentrations of glucose react for 10 min. (B) Fluorescence changes upon the inter-atro

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ction of CdTe QD600 with different concentration of H2O2: (a) before addition ofhe H2O2; (b)–(f) the concentration of the H2O2 is 0.044, 0.11, 0.22, 0.44, 0.66 mMespectively. The inner is the calibration curve corresponds to the optical analysisf different concentrations of H2O2 by the CdTe QDs.

In the glucose detecting system, GOD (GOD-FAD) is used to cat-lyze the oxidation of glucose to release H O and gluconolactone,

2 2esulting in the reductive form of GOD (GOD-FADH2). The catalyticeaction can be depicted as the following:

lucose + GOD-FAD → Gluconolactone + GOD-FADH2 (2)

ig. 4. Schematic principle for assay of H2O2 (A) and glucose (B) based on the elec-ron transfer of QDs and the biochemical reaction.

tronics 26 (2011) 3488–3493 3491

GOD-FADH2 + O2 → GOD-FAD + H2O2 (3)

2H2O2 → 2H2O + O2 (4)

It has been reported that the electron/hole trapped on CdTe QDscan be used as a good electron donor/acceptor. When the H2O2molecule, which obtained from the enzyme reaction, accesses thesurface of the CdTe QDs, the electron-transfer reaction happensimmediately and H2O2 is reduced to O2. Thus the change of thesurface of the CdTe QDs results in quenching of the fluorescenceintensities. As the concentrations of the glucose increases, thebiocatalyzed reaction produces more H2O2 which quenches thefluorescence intensities severely. If only glucose is added into thedetecting system, the fluorescence intensities of QDs do not change(see from Supporting information Fig. S4). Numerous oxidases gen-erate hydrogen peroxide as a product, so the effect of the generatedH2O2 on the fluorescence of the QDs may provide a versatile fluo-rescent reporter for the activities of oxidases and the detection oftheir substrates.

3.5. Analysis of the serum samples

In order to investigate the possible application of this bioana-lytical platform in clinical analysis, the system was also tested inreal human serum. The fluorescence intensity of the QDs no sig-nificantly changed after adding the serum (see from Supportinginformation Fig. S5). The results and the data obtained by Auto-matic Biochemical Analyzer were shown in Table 1. The glucose andLDH in serum was firstly detected by Automatic Biochemical Ana-lyzer, and then 250 �L of serum sample was diluted to 500 �L withPBS solution (pH = 7.0) containing QDs and corresponding reac-tants. The concentration of the glucose and LDH in blood samplewas calculated from the calibration curve. From the Table 1, wecan see a good agreement between the data determined by twomethods. It proved that the detection system ascertained a prac-tical application of the proposed bioanalytical platform in clinicalanalysis.

3.6. Dual simultaneous detection for LDH and glucose

To obtain further application of the QDs-based sensor in thedetection of biomolecules, we also devised the dual assay todetect glucose and the activity of LDH synchronously by microcup(3 × 3 mm) structure on glass. Based on the above results aboutthe relation between the enzyme reactions and the fluorescenceintensities of QDs, we prepared dual-assay by two different sizesof CdTe QDs. Fig. 5A shows that the increment of the enzymes orthe glucose leads to changes in the fluorescence intensities of twokinds of QDs (QD545, QD600), respectively. The fluorescence inten-sities of QD545 (Emission wavelength is 545 nm) are observed tobe intensified by the increment of LDH (Fig. 5A, from QD545-a toQD545-c). On the other hand, the fluorescence intensities of QD600are quenched when the amount of glucose is increased (see fromQD600-a to QD600-c). As mentioned above, the fluorescence inten-sities of the two QDs could be modulated to turn on or turn off todetect the enzyme and glucose synchronously. From the fluores-cence images shown in Fig. 5B, the green fluorescence from QD545(a) is quenched by the NAD (b) then the green fluorescence is inten-sified by adding the LDH and the substrate LL (c). QD600 (d) andQD600 with GOD (e) emit a red color. (For interpretation of the ref-

erences to color in this sentence, the reader is referred to the webversion of the article.) The red fluorescence is quenched (f) in thepresent of glucose. Based on these results, the QDs-based bioana-lytical platform provides a universal way to analyze enzymes andsubstrates synchronously.

3492 L. Yang et al. / Biosensors and Bioelectronics 26 (2011) 3488–3493

Table 1Glucose and LDH concentrations in serum samples tested by the proposed method and Automatic Biochemical Analyzer.

Serum sample Clinical assay concentration Proposed method (n = 3) concentration RSD (%)

Glucose (mM) LDH (U/L) Glucose (mM) LDH (U/L)

Sample 1 5.336 5.446 2.06Sample 2 4.827 4.883 1.16Sample 3 5.157Sample 3 292.37Sample 4 272.69Sample 5 266.51

Fig. 5. (A) Fluorescent spectra of QD545 with the presence of LDH and QD600 withthe different concentrations of glucose. The fluorescence profiles of the two cham-bers were obtained simultaneously. Each curve represents one condition. Curve (1)represents QD545 and QD600 in two chambers of the microcup, respectively; curve(2) represents QD545 + 1 mM NAD and QD600 + 1000 U/L GOD in two chambers ofthe microcup, respectively; curve (3) represents QD545 + 1 mM NAD + 1 mM LL andQD600 + 1000 U/L GOD in two chambers of the microcup, respectively, and thenadded the solution containing 750 U/L LDH and 4.44 mM glucose; curve (4) repre-sents QD545 + 1 mM NAD + 1 mM LL and QD600 + 1000 U/L GOD in two chambers ofthe microcup, respectively, and then added the solution containing 1500 U/L LDHa(LG

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nd 8.33 mM glucose. (B) Fluorescent image of QD545 in the absence (a) or presenceb) of 1 mM NAD, and in the presence of 1 mM NAD, 1 mM LL as well as 1500 U/LDH (c); fluorescent image of QD600 in the absence (d) or presence (e) of 1000 U/LOD, and in the 1000 U/L GOD and 8.33 mM glucose (f).

. Conclusions

In summary, we present a bioanalytical platform based on CdTeDs which provides a novel protocol for selective recognizing of

he activity of the LDH and the glucose synchronously withoutdditional functionalization of QDs. A proposed mechanism is put

orward based on the fluorescence quenching of CdTe QDs, whichs caused by the NAD or H2O2 which is produced from the oxidationf the glucose. In comparison with established enzyme assay, thisioanalytical platform has the significant advantages with simplereparation and high sensitivity in the analysis of the activity of

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NAD-dependent enzyme and the H2O2-generative substrates. Thedetection system is tested the glucose and LDH concentration inhuman serum sample. Furthermore, the detection system based onCdTe QDs provides not only a versatile tool for the analysis by turn-on and turn-off process synchronously, but also the detection ofenzyme and substrate at the same time. Although the developmentof this dual-assay based on QDs is still in its preliminary stages,we believe this method is promising for application of multiplexeddetection in clinical diagnosis.

Acknowledgements

We acknowledge financial support from the National ScienceFoundation of China (60602006, 60736001), National Hi-Tech 863Programme (no. 2007AA021803), and the Knowledge InnovationProgram of the Chinese Academy of Sciences (TYF0808).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bios.2011.01.031.

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