Molecular Beacon Based Bioassay for Highly Sensitive and Selective

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Molecular Beacon Based Bioassay for Highly Sensitive and Selective Detection of Nicotinamide Adenine Dinucleotide and the Activity of Alanine Aminotransferase Zhiwen Tang, Pei Liu, Changbei Ma, Xiaohai Yang, Kemin Wang,* Weihong Tan, and Xiaoyuan Lv State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha 410082, China

bS Supporting Information ABSTRACT: We have developed a new approach to detect nicotinamide adenine dinucleotide (NADþ) with high specificity and sensitivity using molecular beacons (MBs) and employed it in the investigation of NADþ related biological processes, such as calorie restriction and alanine aminotransferase (ALT) activation. The E. coli DNA ligase would catalyze the ligation of two short oligonucleotides that complement with an MB only in the presence of NADþ, resulting in the opening of the MB and the restoration of fluorescent signal. Thanks to the high sensitivity of the MB probe and the fidelity of E. coli DNA ligase toward its substrates, this approach can detect 0.3 nM NADþ with high selectivity against other NADþ analogs. This novel assay can also provide a convenient and robust way to analyze NADþ in biological samples such as cell lysate. As NADþ plays an essential role in many biochemical processes, this method can be used to investigate NADþ related life processes. For instance, the effect of calorie restriction on the intracellular NADþ level in MCF7 cells has been studied using this new assay. Moreover, this approach was also successfully used to analyze the activity of ALT. Therefore, this novel NADþ assay holds wide applicability as an analytical tool in biochemical and biomedical research.

icotinamide adenine dinucleotide, NADþ, a small but important molecule, exists in all life forms and plays a critical role in physiology processes.1 Recent studies revealed some interesting and important characteristics of NADþ. Because of its roles in the regulation of transcriptional pathways and extracellular signaling,2,3 NADþ is considered a putative regulator of gene transcription, cell differentiation, and age-associated diseases such as cancer, diabetes, and neurodegenerative diseases.1-5 Intracellular NADþ level is regarded as an important readout of metabolic status and fluctuates during certain physiological processes or upon environmental stimulations such as calorie restriction.5,6 NADþ participates in many biological processes, including the regulation of energy metabolism, DNA repair, and transcription.1,7 Enzymes that utilize NADþ as a substrate include some NAD-dependent DNA ligases,8 NADdependent oxidoreductases, poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP), (cyclic adenosine diphosphate (cADP)-ribose) synthases, and sirtuins (lysine deacetylases).9 Therefore, sensitive and precise NADþ detection should provide accurate and important information for disease diagnosis and therapy as well as basic studies. Current NADþ detection is based on measuring the absorbance at 340 nm or its fluorescence emission at 450 nm after converting NADþ to reduced nicotinamide adenine dinucleotide (NADH).10 An alternative approach uses an enzymatic cycling assay, which can achieve a detection limit of about 10-13 mol per

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assay by coupling several enzymatic reactions at the cost of additional time-consuming procedures.11-13 In such a cycling assay, the cyclic transform of NADþ/nicotinamide adenine dinucleotide phosphate (NADPþ) and NADH/reduced nicotinamide adenine dinucleotide phosphate (NADPH) enhances the sensitivity but results in poor selectivity. These assays are unable to distinguish NADþ analogs such as NADH, NADPþ, and NADPH and, thus, can not accurately determine NADþ concentration when it is present in a complex sample. In addition, these traditional NADþ assays are often insufficient to provide precise NADþ information for research and clinical applications without complicated sample pretreatment and/or separation procedures. Other methods currently available for the determination of NADþ include fluorescence imaging,14 enzymatic assay,15 high-performance liquid chromatography (HPLC),16 capillary electrophoresis,17 and electrospray ionization mass spectrometry (ESI-MS).18 These methods, however, are either not sensitive enough for biochemical analysis or require expensive equipment and well trained professional operators. Since they were first introduced,19 molecular beacons (MBs) have been broadly used in the fields of biochemistry, biology, biotechnology, and medical research for their excellent sensitivity Received: October 18, 2010 Accepted: February 16, 2011 Published: March 14, 2011 2505

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Scheme 1. Schematic Detection of NADþ Using Molecular Beacon and E. coli DNA Ligasea

a

The underlined sequence in the MB1 indicates the stem sequence. Letter “p” at the 50 end of Oligo2 sequence represents the phosphate.

and specificity.19-23 MBs can be not only a robust tool for DNA/ RNA studies24 but also promising probes for monitoring DNAprotein interactions.25-27 Recently, our group has utilized the advantages of MBs and various enzymes to develop a series of methods for real-time monitoring of nucleic acid ligation,28 phosphorylation/dephosphorylation,29,30 and double-stranded DNA cleavage.31,32 Moreover, novel detection methods for DNA ligase,33 polymerase,34 restriction endonuclease,35 and adenosine triphosphate (ATP)36,37 have been developed. Herein, we developed a convenient, fast, highly sensitive, and selective assay scheme for the detection of NADþ based on MB. The principle of this detection is illustrated in Scheme 1. As shown in the top left of Scheme 1, the assay is composed of a MB, E. coli DNA ligase, and two short oligonucleotides. The MB is designed to bridge the two oligonucleotides with its loop sequence. In the absence of NADþ, an indispensable cofactor for E. coli DNA ligase to form a ligase-adenylate intermediate, the DNA ligation cannot proceed. When NADþ is present, as illustrated in the top right, the ligase will catalyze the ligation process, thereby joining the two short oligonucleotides using the loop of the MB as a template to form a longer DNA strand which is complementary to the entire MB loop sequence. As a result, the MB’s hairpin structure will be disrupted to form a doublestranded DNA with the ligation product, and the quenched fluorescence will be consequently restored. In this way, the presence of NADþ is efficiently and specifically converted into fluorescence signals, which can be easily and sensitively analyzed. To demonstrate the potential of NADþ detection using MB in biomedical studies and clinical applications, the effect of calorie restriction on the intracellular NADþ level of human cells was investigated. Furthermore, a new assay for the activity of alanine aminotransferase (ALT) was developed on the basis of this novel NADþ assay.

’ EXPERIMENTAL SECTION NADþ Assay Solution. The E. coli DNA ligase used for NADþ

detection was purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. All other chemicals were purchased from Sigma-Aldrich Co. Two molecular beacons were synthesized with the same DNA sequence but different dyes at the 50 terminus. MB1 was

labeled with fluorescein isothiocyanate (50 -(FITC)-CCTCTCCGTGTCTTGTACTTCCCGTCAGAGAGG-(DABCYL)-30 ) for fluorescence imaging, and MB2 used TMR (tetramethylrhodamine) as the fluorophore. MB1, MB2, Oligo1 (50 -GAC GGG AAG-30 ), and Oligo2 (50 -p-TAC AAG ACA C-30 ) were synthesized by TaKaRa Biotechnology (Dalian) Co., Ltd. The NADþ assay buffer was composed of 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM dithiothreitol (DTT), 2.5 mM CaCl2, and 0.05% BSA. All samples were prepared in a 80 μL aliquot of solution containing 250 nM MB2, 250 nM Oligo1, 250 nM Oligo2, and 6.0 units of E. coli DNA ligase for all NADþ assay experiments. The solutions in the experiments were prepared with ultrapure water (Milli-Q 18.2 MΩ 3 cm, Millipore System Inc.). Fluorescent Imaging. A 200 μL NADþ assay buffer solution was prepared in a 200 μL PCR tube to contain 500 nM MB1, 600 nM Oligo1, 600 nM Oligo2, and 10 units of E. coli DNA ligase. The images of the PCR tube were taken with an EDAS 290 (Kodak) imaging cabinet while the tube was excited by a UV transilluminator (UltraLum Inc.). After taking the first photo of the PCR tube containing the assay solution, NADþ was introduced into the tube at the final concentration of 400 nM. Another photo was then taken in 5 min after mixing NADþ with the assay solution. Fluorescence Measurement. All fluorescence measurements of samples were carried out on a F2500 (Hitachi, Japan) with excitation at 521 nm and emission at 578 nm for TMR labeling at the 50 end of the MB2 as a fluorophore. Each sample was incubated at 37 °C for about 8 min, and a steady state was reached before the addition of DNA ligase. The sample was then incubated until the fluorescence again reached equilibrium, after which NADþ, NADþ analog, or cell lysis was introduced into the solution and stirred for 4 s. The fluorescence was recorded synchronously, and the NADþ level was represented by the initial enhancement rate of fluorescence intensity. The initial enhancement rate of fluorescence intensity was determined by the average of fluorescence enhancement rate in 200 s after the addition of E. coli DNA ligase. Specificity Evaluation. The selectivity test was carried out by measuring and comparing the response of NADþ to that of its analogs including NADH, NADPþ, NADPH, ATP, deoxyadenosine triphosphate (dATP), ADP, and adenosine 5'-monophosphate (AMP). The assay solution composition 2506

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Analytical Chemistry and protocol are identical to the previous NADþ assay, and the final concentration of analytes was 40 nM. The response of analyte was recorded, and the initial enhance rate was used to evaluate the specificity of the MB based NADþ assay. Cell Culture and Sampling for Calorie Restriction Experiment. MCF7 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen Corporation) containing different concentrations of glucose (4.5, 2.75, and 1.0 g/L), which simulated the normal and calorie restriction conditions. The MCF7 cells were first treated with 0.5% trypsin (Amresco) to become detached from the flask, after which they were split into 6 well cell culture clusters and cultured overnight in media containing 2.75 g/L glucose. After the MCF7 cells were attached to the plate, the culture media was washed away and replaced with fresh media containing 4.5, 2.75, and 1.0 g/L glucose. Three batches of MCF7 cells cultured with different glucose levels were harvested at half hour intervals after being incubated with fresh media. The cells were transferred into a 1.5 mL eppendoff tube after treatment with 0.5% trypsin (Amresco). The cell numbers were counted three times for accurate quantitation. The cells were washed with 1.0 mL of PBS buffer and spun at 3000 rpm in a centrifuge (Microfuge 18, Beckman) for 5 min, after which the supernatant was removed. After three washes, the cells in each tube were mixed with 50 μL of lysis buffer (150 mmol/L NaCl, 1.0% Tritonx-100, 50 mmol/L Tris-HCl, 1 mmol/L EDTA, 1 mmol/L PMSF) and placed on ice for 30 min to facilitate the lysis process. The lysate was deactivated by heating at 95 °C for 5 min to eliminate the potential interference to the assay system. According to cell number quantitation, a calculated volume of lysate containing 1  104 cells was harvested for the NADþ assay. Assay of ALT. A 40 μL solution of various concentration of ATL (in Tris-HCl buffer) was added to the 150 μL buffer solution 1 consisting of Tris-HCl (100 mM), L-alanine (333 mM), NADH (80 μM), and LDH (0.5 kU/L), pH = 7.4, mixed thoroughly, and incubated at 37 °C for 5 min. Then, 50 μL of solution 2 consisting of Tris-HCl (100 mM), L-alanine (999 mM), and R-ketoglutarate (60 mM) as initiating agent was quickly added into the incubated solution. After 2 min, 2 μL of solution was transferred into the above-mentioned assay solution and the fluorescence signal of the sample was monitored in real time.

’ RESULTS AND DISCUSSION Design and Feasibility of MB Based NADþ Assay. To

demonstrate the feasibility of this approach, a NADþ assay solution was prepared in a 200 μL centrifuge tube containing MB1, Oligo1, Oligo2, and E. coli DNA ligase in the NADþ assay buffer. Two images of the sample were taken with an EDAS 290 imaging cabinet, while the tube was excited by a UV transilluminator, and are shown in Scheme 1. The image on the left shows that the fluorescence was weak in the absence of NADþ, while the right image was taken 5 min after the introduction of NADþ at 400 nM final concentration. The fluorescence intensity of the latter was considerably stronger than the former, indicating the opening of the MB in the presence of NADþ. This result was consistent with the proposed mechanism of the NADþ assay shown in Scheme 1. The presence of NADþ molecule can be efficiently and quickly converted into the opening of MB1, which leads to enhanced fluorescence signal for analysis. Compared with the traditional spectrometric NADþ assays, MB provides

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Figure 1. (A) Fluorescence time courses of samples with addition of different amounts of NADþ. The final concentration of NADþ ranges from 0 to 300 nM: (a) 300, (b) 200, (c) 80, (d) 40, (e) 30, (f) 10, (g)5.0, (h) 0.5, (i) 0.3, and (j) 0 nM. The inset is the time courses of three low concentration samples and control samples. (B) Initial velocity calibration curve. Inset is the calibration curve for concentrations ranging from 0.3 to 40 nM. Measurements were performed in a Tris-HCl buffer (50 mM, pH = 8.0, 10 mM MgCl2, 5 mM DTT, 2.5 mM CaCl2, and 0.05% BSA). All samples were prepared in a 80 μL aliquot of solution containing 250 nM MB2, 250 nM Oligo1, 250 nM Oligo2, and 6.0 units of E. coli DNA ligase (error bars were deduced from N = 3 experiments).

highly sensitive signal and flexible fluorophore labeling; thus, the sensitivity and adaptability of the NADþ assay can be improved by taking advantages of such features of MB. Detection of NADþ. To further characterize the detection range of this assay, a series of samples containing different concentrations of NADþ have been tested. All samples were prepared in an 80 μL aliquot of ligation buffer containing MB2, Oligo1, Oligo2, and E. coli DNA ligase. The fluorescence time courses, after the addition of NADþ to final concentrations ranging from 0 to 300 nM, are plotted in Figure 1A. The initial velocity of the fluorescence change increased as increasing amounts of NADþ were added. The initial velocity is plotted versus NADþ concentration in the range from 0.3 to 300 nM, as shown in Figure 1B. The inset of Figure 1B reveals that the initial velocity is linearly depended on the NADþ concentration in a range from 0 to 40 nM (R2 = 0.999). A detection limit of 0.3 nM (S/N = 3) was achieved, which is about 1 order of magnitude lower than that of cycling assays. The total time of the NADþ assay was less than 4 min, which is faster than traditional cycling assays employing time-consuming procedures. High Selectivity. One of the major problems with the conventional NADþ assay is the interference from a list of other small molecules. The 340 nm absorbance assay suffers from low sensitivity and the overlapping absorbance with other substances 2507

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Figure 2. Selectivity of the MB based NADþ assay. The final concentration of all analytes is 40 nM. The responses are evaluated on the basis of the initial enhancement rate. All data are normalized to the NADþ response. Inset: the response of NADþ analogs presented with smaller scale.

existing in common biological samples. For the enzymatic cycling assay, the sensitivity is improved at the cost of poor selectivity between NADþ and its analogs such as NADPþ and NADH. In addition, complex and time-consuming separation treatments are inevitable when biological samples are analyzed using traditional NADþ assays, limiting the applicability and increasing the cost of the NADþ assay. The selectivity of the MB based NADþ assay was evaluated by comparing the response of NADþ to that of NADH, NADPþ, NADPH, ATP, dATP, ADP, and AMP. These molecules are analogs of NADþ and widely exist in almost all biological samples. As a result, the coexistence of NADþ and its analogs often causes interference in conventional NADþ assays. In a specificity experiment, the response of analytes was represented by the initial rate of fluorescence enhancement calculated from the fluorescence intensity time course and normalized to NADþ response. As shown in Figure 2, all NADþ analogs, including NADH, showed less than 1.5% of the signal produced by NADþ. Compared to current NADþ analyses, this new approach possesses excellent selectivity due to the extreme fidelity of E. coli DNA ligase for NADþ.38 Furthermore, to avoid the background fluorescence from biological or clinical sample matrix, the fluorophore linked to the MB can be easily altered as needed. Overall, the results suggest that this novel assay should be well suited for selective and sensitive detection of NADþ in complex samples such as cell lysates. NADþ Assay for Cell Lysate Analysis. In practice, NADþ assays are carried out with biological samples containing complex biological and chemical matrixes. For traditional NADþ analysis, the pretreatment and separation of samples are inevitable in order to eliminate the interferences of other substances existing in real samples. The high selectivity of this novel NADþ assay offers the potential to analyze the biological samples without the time-consuming and tedious pretreatments. To affirm this point, the effect of cell lysate alone on the fluorescence of assay solution was investigated. After the deactivation at 90 °C for 5 min, the cell lysate did not show any disturbance (data not shown). To further validate this assay using cell lysate sample, the standard addition method was employed to test cell lysate samples. A series of samples containing 104 cells lysate with additional NADþ ranging from 0 to 20 nM were tested, and the initial fluorescence enhancement rates were plotted as a function of the concentration of added NADþ in Figure 3. The relationship between the initial rate and added NADþ is linear, and the NADþ concentration of lysate sample determined by the standard addition method is 18.4 ( 0.3 nM. Using the new

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Figure 3. Standard addition plot of NADþ assay in cell lysate. The absolute value of the x-intercept is 18.2 nM.

Figure 4. Effects of calorie restriction on the intracellular NADþ in MCF-7 cells cultured in three media with different concentrations of glucose (error bars were deduced from N = 3 experiments).

method developed in this paper, the NADþ concentration in lysate sample is 17.8 ( 1.1 nM. The t test shows that the t-value is 0.74, which is smaller than t0.95,2 (4.3), so the difference between results of two assays is not significant. These results demonstrated that this novel approach worked well with the cell lysate sample. Effect of Calorie Restriction on the Intracellular NADþ Level in MCF7 Cells. NADþ detection is important in many biomedical applications such as calorie restriction study. Calorie restriction is a dietary regimen which has been known to extend the life span and mitigate a wide spectrum of diseases.6 However, the molecular basis of calorie restriction remains unsolved. Recent studies indicated that the Sir2 gene product might mediate the calorie restriction effect.5,6,9 As a result of the metabolic shift toward respiration during calorie restriction, the intracellular NADþ level would increase and activate the Sir2.34 Recently, there has been efforts in revealing the mechanism of calorie restriction effect on mammal cells, but the role of the intracellular NADþ level was not discussed due to difficulties in its precise quantitation.4 To determine the calorie restriction effect on the intracellular NADþ level in human cells, the MCF7 cell was selected as a model and cultured in Dulbecco’s modified Eagle’s medium (Invitrogen Corporation) containing different concentrations of glucose (4.5, 2.75, and 1.0 g/L). The intracellular NADþ levels were assayed by measuring whole cell extractions of 104 MCF7 cells at half hour intervals. The data plotted in Figure 4 show that the intracellular NADþ levels increased while the glucose 2508

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Scheme 2. Schematic Detection of ALT Using Molecular Beacon and E. coli DNA Ligasea

a

The molecular beacon is labeled with a TMR (solid circle) and a Dabcyl (solid square). The two short curves correspond to perfect complementary oligo A and oligo B. NMN: Nicotinamide mononucleotide. AMP: adenosine monophosphate.

concentration dropped in the culture media. The NADþ level of MCF7 cultured with 1.0 g/L glucose media is about 28% higher than that of the cells grown in 4.5 g/L glucose media. The P-values for each time point from 0.5 to 2 h are 0.011, 0.001, 0.004, and 0.001, respectively, which means that the NADþ level alteration under calorie restriction is significant. This result suggests a metabolic shift associated with calorie restriction, which has been proposed to explain the calorie restriction effect at the cellular level.39 Assay of ALT Activity. In biomedical research and clinical applications, a number of important bioassays involve NADþ/ NADH consuming enzymatic processes of which ALT is a classical example.40 ALT is found in human serum and various tissues and mostly associated with liver. Significantly elevated levels of ALT often suggest the existence of medical problems such as viral hepatitis, congestive heart failure, liver damage, bile duct problems, infectious mononucleosis, and myopathy.41 Therefore, the ALT assay is important in diagnosis and treatment of these diseases. On the basis of the aforementioned NADþ analysis strategy, we developed a novel ALT assay. As shown in Scheme 2, ALT catalyzes the transfer of an amino group from alanine to R-ketoglutarate, producing pyruvate and glutamate. Subsequently, lactate dehydrogenase (LDH) mediates the oxidation of pyruvate, yielding lactate acid and NADþ. As a result, the amount of ALT can be determined by measuring the concentration of NADþ, which can be precisely quantified using the MB based NADþ assay. To investigate the feasibility of this proposed assay, various amounts of ALT was introduced and the fluorescence of samples was recorded in real-time, as shown in Figure 5A. Figure 5B represents the relationship between the initial velocity of fluorescence enhancement and ALT concentration. Clearly, the fluorescence initial velocity increased continuously as a function of ALT concentration in the range up to 50 U/L. The fitting equation of the curve shown in Figure 5B is half of a hyperbola, which is a typical fitting mode for an enzyme assay: y ¼ 0:0760 þ ½0:474/x=ð9:35 þ xÞ where y is the initial velocity of fluorescence enhancement and x is the final concentration of ALT. The inset of Figure 5B shows that the initial velocity is linearly dependent on the ALT concentration in a linear range up to 3.13 U/L (R2 = 0.971) with a detection limit of 0.25 U/L (S/N = 3). The ALT level in normal human serum is 10-40 U/L reported by most laboratories.42 Using a simple dilution of serum as the sample preparation step, this MB and E. coli. ligase based ALT assay

Figure 5. (A) Kinetic fluorescence-time curves for the ALT sensing assay. Concentration of ALT in samples ranges from 0 to 50 U/L: (a) 50.0, (b) 25.0, (c) 12.5, (d) 6.25, (e) 3.13, (f) 1.56, (g) 0.78, and (h) 0 U/L. (B) Initial velocity calibration curve. Inset is the calibration curve for ALT concentration ranging from 0 to 3.13 U/L. The measurement conditions are the same as in Figure 1 (error bars were deduced from N = 3 experiments).

could be suitable for the detectable range of human serum samples. The success of this new assay of ALT clearly demonstrates that MB based NADþ assay can be readily adopted to detect NADþ/NADH consuming enzymatic processes, which are critical in many biological and biomedical studies.

’ CONCLUSION In conclusion, using MBs for the detection of small molecules such as NADþ represents a novel class of biomolecular assays. Compared to traditional NADþ assays, the MB based NADþ assay is fast, highly sensitive, and selective without complicated treatments, which make it valuable for analyzing complex biological samples. As demonstrated in the current report, this new assay opens up new possibilities in the use of MBs to study life processes involving NADþ, such as the effect of calorie restriction on intracellular NADþ level. Moreover, it can be utilized to detect NADþ/NADH consuming enzymatic processes by coupling it with relevant enzymatic reactions, as demonstrated in the ALT activity assay. In addition, the principle of these novel MB based assays for NADþ and relevant enzymes can be used to construct reusable optical or electrochemical biosensors by immobilizing MBs on the sensor surface. Altogether, these novel MB based assays hold potential for broad applications in biochemical and biomedical study, as well as in clinic applications. 2509

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’ ASSOCIATED CONTENT

bS

Supporting Information. Figure of the optimization of LDH concentrations. This material is available free of charge via the Internet at http://pubs.acs.org.

Notes

Zhiwen Tang and Pei Liu contributed equally to this work.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: þ86-731-88821566. Fax: þ86-731-88821566.

’ ACKNOWLEDGMENT This work was supported in part by the Key Project of Natural Science Foundation of China (90606003), International Science and Technology Cooperation Program of China (2010DFB30300), Program for New Century Excellent Talents in University (NCET09-0338), and Hunan Provincial Natural Science Foundation of China (08JJ1002, 10JJ7002). We thank Dr. Zehui Cao for his helpful discussion and suggestions. ’ REFERENCES (1) (a) Lin, S.-J.; Guarente, L. Curr. Opin. Cell Biol. 2003, 15, 241–246. (b) Canto, C.; Gerhart-Hines, Z.; Feige, J. N.; Lagouge, M.; Noriega, L.; Milne, J. C.; Elliott, P. J.; Puigserver, P.; Auwerx, J.. Nature 2009, 458, 1056–1060. (2) Araki, T.; Sasaki, Y.; Milbrandt, J. Science 2004, 305, 1010–1013. (3) Kawamura, H.; Aswad, F.; Minagawa, M.; Malone, K.; Kaslow, H.; Koch-Nolte, F.; Schott, W. H.; Leiter, E. H.; Dennert, G. J. Immunol. 2005, 174, 1971–1979. (4) Cohen, H. Y.; Miller, C.; Bitterman, K. J.; Wall, N. R.; Hekking, B.; Kessler, B.; Howitz, K. T.; Gorospe, M.; de Cabo, R.; Sinclair, D. A. Science 2004, 305, 390–392. (5) Guarente, L.; Picard, F. Cell 2005, 120, 473–482. (6) Koubova, J.; Guarente, L. Genes Dev. 2003, 17, 313–321. (7) Ying, W. Front. Biosci. 2007, 12, 1863–1888. (8) Wilkinson, A.; Day, J.; Bowater, R. Mol. Microbiol. 2001, 40, 1241–1248. (9) Belenky, P.; Bogan, K. L.; Brenner, C. Trends Biochem. Sci. 2007, 32, 12–19. (10) Henry, R. J. Clin. Chem.: Principle Tech. 1968, 664–666. (11) Matsumura, H.; Miyachi, S. Methods Enzymol. 1980, 69, 465–470. (12) Bernofsk, C.; Swan, M. Anal. Biochem. 1973, 53, 452–458. (13) Gibon, Y.; Larher, F. Anal. Biochem. 1997, 251, 153–157. (14) Kasischke, K. A.; Vishwasrao, H. D.; Fisher, P. J. Science 2004, 305, 99–103. (15) Dekoning, W.; Vandam, K. Anal. Biochem. 1992, 204, 118–123. (16) (a) Yamada, K.; Hara, N.; Shibata, T. Anal. Biochem. 2006, 352, 282–285. (b) Xie, W.; Xu, A.; Yeung, E. S. Anal. Chem. 2009, 81, 1280–1284. (17) Xie, W.; Xu, A.; Yeung, E. S. Anal. Chem. 2009, 81, 1280–1284. (18) Sadanaga-Akiyoshi, F.; Yao, H.; Tanuma, S. Neurochem. Res. 2003, 28, 1227–1234. (19) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303–308. (20) Tan, W.; Wang, K.; Drake, T. J. Curr. Opin. Chem. Biol. 2004, 8, 547–553. (21) Fang, X.; Liu, X.; Schuster, S.; Tan, W. J. Am. Chem. Soc. 1999, 121, 2921–2922.

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