Single Quantum Dot Based Nanosensor for Renin Assay - Analytical

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Single Quantum Dot Based Nanosensor for Renin Assay Yi Long,† Ling-fei Zhang,†,‡ Yan Zhang,† and Chun-yang Zhang*,† †

Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China ‡ Weihai Municipal Center for Disease Control and Prevention, Shandong 264200, China ABSTRACT: Evaluation of plasma renin activity is essential to the assessment of renin-related diseases such as hypertension, congestive heart failure, and cancers. Here, we develop a single quantum dot (QD) based nanosensor for sensitive detection of renin activity. This single-QD-based nanosensor consists of a streptavidin-coated QD and multiple biotinylated and Cy5-labeled peptide substrates, which form a QD/substrate/Cy5 complex where fluorescence resonance energy transfer (FRET) occurs with the QD as the donor and Cy5 as the acceptor. The presence of renin leads to the cleavage of the substrate and the separation of Cy5 from the QD and consequently the decrease of FRET efficiency and the reduction of Cy5 counts. Through the measurement of Cy5 counts by total internal reflection fluorescence (TIRF) microscopy, the renin activity can be quantitatively evaluated at the single-molecule level. This single-QD-based nanosensor can measure not only the renin concentration, but also the enzymatic velocity and the Michaelis−Menten kinetic parameters, and has significant advantages of simplicity, low cost with minimum sample consumption, and high sensitivity with a detection limit of 25 pM. This single-QD-based nanosensor might be further applied to monitor a variety of important enzymatic biomarkers such as kinases and endonuleases.

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the direct and continuous measurement of renin activity.10 The reported intramolecularly quenched fluorogenic substrates employ 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS)/4-(4-dimethylaminophenylazo) benzoic acid (DABCYL), 18 anthranilic acid/N-(2,4-dinitrophenyl)ethylenediamine (EDDnp),19 and 7-methoxycoumarin/2,4dinitrophenyl (DNP)10 as the fluorophore/quencher pairs to measure the renin activity. Nevertheless, the organic dye- and protein-based fluorophores have some chemical and photophysical disadvantages, such as the susceptibility to pH changes and photobleaching, poor aqueous stability, narrow absorption windows with broad emission spectra, and small Stokes shifts.20,21 Thus, the development of reliable and sensitive methods for renin assay still remains a great challenge. The quantum dot (QD) has emerged as a viable alternative to the organic fluorophores in genomic analysis, immunoassay, in vitro and in vivo imaging, and drug delivery.22−26 The QD has significant advantages of good resistance to chemical and photodegradation, large absorption cross sections over a broad range of excitation wavelengths, size-dependent emission spectra with narrow bandwidths, relatively high quantum yield, and a stable surface for further chemical modification.27−30 Recently, the QD has been used as the fluorescence resonance energy transfer (FRET) donor in the biosensing applications for sensitive detection of nucleic acids, proteins, and small molecules.31−34 The use of the QD as the FRET

enin plays an important role in the renin−angiotensin system (RAS) which regulates blood pressure and electrolyte homeostasis.1 As the first and rate-limiting step of RAS activation, renin releases from the juxtaglomerular cells of the kidney and initiates the enzyme cascade.2−4 Renin can hydrolyze angiotensinogen from the liver into angiotensin I,5 and angiotensin I may be further converted into angiotensin II in the lung by endothelial-bound angiotensin-converting enzyme.6−8 Angiotensin II is one of the most potent vasopressors that ultimately integrates the functions of cardiovascular and renal systems in the control of blood pressure.9 Recent researches indicate that excessive secretion of renin may cause not only hypertension but also congestive heart failure and cancers.10,11 Therefore, the development of simple and sensitive methods for renin assay is essential to the early clinical diagnosis and the discovery of renin inhibitors for the treatment of renin-related diseases. So far, a variety of methods have been developed for renin assay.10 Among them, radioimmunoassay provides an indirect way for the detection of plasma renin activity through analyzing angiotensin I generated by renin, but it suffers from long incubation time and the inability to reflect the real concentration of active renin.12,13 Monoclonal antibody-based radioimmunometric assay offers a direct way for renin assay.14 However, the radioimmunoassay is very complicated, timeconsuming, and at the risk of radioactive material-related pollution. Alternatively, several fluorescence methods have been developed on the basis of dye-labeled substrates,15−17 but these methods are usually discontinuous with a low throughput.10 The intramolecularly quenched fluorogenic substrates enable © 2012 American Chemical Society

Received: August 8, 2012 Accepted: September 24, 2012 Published: September 24, 2012 8846

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50 μg/mL BSA, 1 mg/mL Trolox. An oxygen-scavenging buffer (1 mg/mL glucose oxidase, 0.04% mg/mL catalase, and 0.4% (w/v) D-glucose) was included in the imaging buffer to minimize the photobleaching. The QDs were excited by a Jive 488 nm DPSS laser (Cobolt) via total internal reflection. The fluorescence signals from both the QD donor and the Cy5 acceptor were collected by an oil immersion objective (NA 1.45, 100×, Olympus), separated by Optosplit II image splitter (Cairn Research), and imaged onto the two halves of an Andor Ixon DU897 EMCCD with a time resolution of 50 ms. The reaction solution with the final concentration of 20 pM QDs was added to the imaging chamber for single-molecule measurement. Data Analysis. Data analysis was performed using Image J (version 1.4.3.67, Broken Symmetry Software, U.S.A.). An imaging region of 200 × 450 pixels was selected for data analysis. The Cy5 counts were obtained by calculating 10 frames with the “analyze particles” function of Image J. For the evaluation of detection sensitivity, the reduction of Cy5 counts was calculated based on eq 1:

donor offers several unique spectroscopic properties unmatched in any available organic fluorophores, including improved FRET efficiency as a result of coupling multiple acceptors around a single QD, tunable spectral overlap between the QD and the acceptor, minimization of direct acceptor excitation, and multiplex FRET configurations.35−37 Due to its unique capability of detecting a single molecule with a high signal-to-noise ratio, the single-molecule detection technique has become a powerful tool in the biological and biomedical researches.38,39 The single-molecule detection technique coupled with microdroplets,40,41 capillary and microchannels,42,43 hydrodynamically focused flows,44 nanometer-scale pores,45 confocal fluorescence microscopy,46 epifluorescence microscopy,47,48 and total internal reflection fluorescence (TIRF) microscopy49,50 has been employed for sensitive detection of various biomolecules. Recently, the single-molecule detection technique in combination with the QD has been used to sensitively detect DNA, miRNA, and cocaine.51−53 However, the detection of protein, especially the enzyme, using the single-molecule detection technique in combination with the QD has never been reported so far. Herein, we develop a single-QD-based nanosensor for sensitive detection of renin activity on the basis of the singlemolecule detection technique in combination with the QD. This single-QD-based nanosensor consists of a streptavidincoated QD and multiple biotinylated and Cy5-labeled peptide substrates, which form a QD/substrate/Cy5 complex where FRET occurs with the QD as the donor and Cy5 as the acceptor. In the presence of renin, the peptide substrate is cleaved, resulting in the separation of Cy5 acceptor from the QD donor and consequently the decrease of FRET efficiency and the reduction of Cy5 counts. Through the measurement of Cy5 counts by TIRF microscopy, the renin activity can be quantitatively evaluated at the single-molecule level. We further demonstrate that this single-QD-based nanosensor can measure not only the renin concentration but also the enzymatic velocity and the Michaelis−Menten kinetic parameters.

Nreduction = Nnegative − Nrenin

(1)

where Nreduction is the reduction of Cy5 counts, Nnegative is the Cy5 counts from the negative control in the absence of renin, and Nrenin is the Cy5 counts in the presence of renin.

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RESULTS AND DISCUSSION Principle of the Single-QD-Based Nanosensor for Renin Assay. The strategy for renin assay is shown in Scheme 1. The single-QD-based nanosensor consists of a streptavidinScheme 1. Schematic Illustration of the Single-QD-Based Nanosensor for Renin Assaya

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MATERIALS AND METHODS Materials. The human renin was purchased from Molecular Innovations, Inc. (Beijing, China). The specific substrate was synthesized and purified by Chinese Peptide Co. (Hangzhou, China), and its sequence is biotin-Lys-His-Pro-Phe-His-LeuVal-Ile-His-Lys-Cy5. The inhibitor CGP-29287 (Z-Arg-ArgPro-Phe-His-Sta-Ile-His-Nepsilon-Boc-Lys methyl ester) was ordered from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Streptavidin-coated 605 nm emission QDs were obtained from Invitrogen Co. (Carlsbad, CA, U.S.A.). Renin Proteolytic Assay. The renin proteolytic reaction was carried out at 37 °C in 20 μL of reaction buffer containing 50 mM MOPS/NaOH (pH 7.0), 2 mM EDTA, 0.5% BSA, 0.1% NaN3, 1% DMSO, and different concentrations of renin and the substrate. After the reaction, 1 μL of 2 μM QDs and the corresponding buffer (Invitrogen Co., Carlsbad, CA, U.S.A.) were added into the reaction solution to reach a final volume of 100 μL. The substrates and the QDs were incubated at room temperature to make streptavidin-coated QDs conjugate to biotinylated substrates. Before single-molecule FRET measurement, the glass slides were pretreated according to the method developed by Chan et al.54 In the single-molecule measurement, 100 μL of reaction solution was further diluted 1000-fold with the buffer containing 67 mM glycine−KOH (pH 9.4), 2.5 mM MgCl2,

In the absence of renin, Cy5 fluorescence signal is observed due to FRET between the QD donor and Cy5 acceptors in the QD/ substrate/Cy5 complex. While in the presence of renin, FRET will disappear due to the cleavage of the substrates by renin and the dissociation of Cy5 acceptors from the QD donor.

a

coated 605 nm emission QD and multiple biotinylated and Cy5-labeled peptide substrates. In the absence of renin, biotinylated and Cy5-labeled peptide substrates assemble on the surface of the QD through specific biotin−streptavidin interaction, forming a QD/substrate/Cy5 complex. The Förster distance (R0) of the 605 nm emission QD/Cy5 pair, i.e., the distance at which the energy transfer efficiency is 50%, is 8847

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calculated to be 69.4 Å, and FRET efficiency (E) is calculated based on eq 2:55 E=

nR 0 6 nR 0 6 + r 6

(2)

where n is the average number of acceptor molecules interacting with one donor and r is the average donor− acceptor separation distance. Consequently, the assembling of multiple Cy5-labeled substrates on the surface of a single QD will significantly improve the FRET efficiency. Due to the efficient FRET between the QD donor and Cy5 acceptor in the QD/substrate/Cy5 complex, Cy5 fluorescence signal is observed with the corresponding decrease in the QD fluorescence signal as a result of the energy transfer from the QD to Cy5. While in the presence of renin, renin will recognize and hydrolyze the peptide substrate at the specific bond between Leu and Val,10 leading to the separation of Cy5 acceptors from the QD donor and consequently the decrease of FRET efficiency and the reduction of Cy5 counts. Through the measurement of Cy5 counts by TIRF microscopy, the renin activity can be quantitatively evaluated at the single-molecule level. In this research, TIRF microscopy was used for the imaging of the single-QD-based nanosensor. TIRF is a technology that allows selective excitation of fluorescent molecules within ∼100 nm of the coverslip.56 In theory, the distance between the adjacent amino acids in peptide is ∼8.3 Å at most,57 and the total length of peptide substrate used in this research is estimated to be ∼83 Å. On the basis of the theoretical calculation, the radius of a streptavidin-coated 605 nm emission QD is ∼75 Å.55 As a result, the largest separation distance between the QD donor and Cy5 acceptor is calculated to be ∼23.3 nm, which is within the excitation field of TIRF.56 In fact, due to the heterogeneous structures and orientation of peptide, the Cy5 acceptors are always in close proximity or tangential to the QD surface,58 leading to efficient FRET between the QD donor and Cy5 acceptors in the QD/ substrate/Cy5 complex. Figure 1 shows the fluorescence images of the single-QDbased nanosensor in the presence (Figure 1, parts A and B) and in the absence (Figure 1, parts C and D) of renin obtained by TIRF microscopy. In the presence of 5 nM renin, only the QD fluorescence signals (Figure 1A) are observed, but no Cy5 fluorescence signal (Figure 1B) is observed, indicating that the substrates are all proteolyzed by 5 nM renin within 120 min. The disappearance of Cy5 fluorescence signal can be explained by the separation of all Cy5 acceptors from the QD donor due to the cleavage of the peptide substrates by renin. In contrast, both the QD (Figure 1C) and Cy5 fluorescence signals (Figure 1D) are observed in the negative control without renin, indicating the efficient FRET between the QD donor and Cy5 acceptors in the QD/substrate/Cy5 complex. The peptide substrate can be cleaved by renin at the hydrolyzed bond between Leu and Val,10 resulting in the separation of Cy5 from the QD and the decrease of FRET efficiency; thus, the reduction of Cy5 counts is a direct evidence of the peptide cleavage and can be used to evaluate the renin activity. It is worth noting that the QD fluorescence signals in the presence of renin (Figure 1A) are much brighter than those in the absence of renin (Figure 1C). This can be explained by the quenching of the QD fluorescence as a result of efficient energy

Figure 1. Fluorescence images of the single-QD-based nanosensor in the presence (A and B) and in the absence (C and D) of renin obtained by TIRF microscopy. (A and B) Fluorescence images of the QD (A) and Cy5 (B) in the presence of 5 nM renin. (C and D) Fluorescence images of the QD (C) and Cy5 (D) in the absence of renin. The scale bar is 4 μm. The substrate concentration is 4.8 μM, and the QD concentration is 0.1 μM in the reaction solution.

transfer from the QD donor to Cy5 acceptors in the QD/ substrate/Cy5 complex (Figure 1, parts C and D). Influence of Substrate-to-QD Ratio on FRET Efficiency. To determine the influence of the Cy5-labeled substrate-to-QD ratio on FRET efficiency, both the singleQD-based nanosensor and the bulk measurement were investigated. In the single-QD-based nanosensor, the FRET efficiency (E) is calculated based on eq 3:59 E=1−

∑ IDA FDA =1− ∑ ID FD

(3)

where ∑IDA is the sum of QD fluorescence intensities in the presence of Cy5 acceptors, and∑ID is the sum of QD fluorescence intensities in the absence of Cy5 acceptors. As shown in Figure 2A, both the FRET efficiency and the Cy5 counts increase with the increasing Cy5-labeled substrate-toQD ratio from 1/1 to 48/1; but beyond the ratio of 48/1, there is no further increase in either the FRET efficiency or the Cy5 counts, indicating that the biotin-binding sites on the surface of the QD have been saturated. It should be noted that a good linear correlation is obtained between the Cy5 counts and the Cy5-labeled substrate-to-QD ratio in the range from 1/1 to 48/ 1 (red line in Figure 2A). The obtained Cy5-labeled substrateto-QD ratio of 48:1 is very close to the theoretically calculated biotin-binding sites per QD of 45. Because there are three available biotin-binding sites per streptavidin after conjugation to the QD, and each QD is conjugated with 12−15 streptavidins,60 in theory, there are up to 45 biotin-binding sites per QD. The minor discrepancy between the obtained Cy5-labeled substrate-to-QD ratio and the theoretically calculated biotin-binding sites per QD might be attributed to 8848

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fluorescence emission and the distortion of band shape due to the reabsorption of emitted radiation,61 resulting in the decrease of Cy5 fluorescence intensity beyond the ratio of 48/1 in the bulk measurement. Therefore, the single-QD-based nanosensor is more accurate than the bulk measurement. In order to obtain a broad dynamic range, the Cy5-labeled substrate-to-QD ratio of 48/1 is used in the following research. Time Profile of Renin Activity. We further investigated the time profile of renin activity using the single-QD-based nanosensor. The renin activity was terminated by adding 5 nM inhibitor with either a time interval of 10 min before 60 min or a time interval of 30 min after 60 min. Previous research demonstrated that the enzymatic cleavage rate of a substrate with a QD as the donor was much lower than that with a molecule as the donor.23 To avoid the adverse effect of QDinduced steric hindrance upon the substrate−renin reaction, the QDs were added into the solution to form the QD/substrate/ Cy5 complexes after the termination of reaction. Figure 3

Figure 2. (A) Variance of FRET efficiency (○) and Cy5 counts (●) as a function of the Cy5-labeled substrate-to-QD ratio in the single-QDbased nanosensor. (B) Variance of FRET efficiency (○) and Cy5 fluorescence intensity (●) as a function of the Cy5-labeled substrateto-QD ratio in the bulk measurement. Error bars show the standard deviation of three experiments.

the difference in the synthesis of streptavidin-conjugated QD from batch to batch. In the bulk measurement, the FRET efficiency (E) is calculated based on eq 4:59

E=1−

FDA FD

Figure 3. Variance of Cy5 counts as a function of reaction time in the presence (●) and in the absence (○) of 5 nM renin. The substrate concentration is 4.8 μM, and the QD concentration is 0.1 μM in the reaction solution. Error bars show the standard deviation of three experiments.

(4)

where FDA is the QD fluorescence intensity in the presence of Cy5 acceptors and FD is the QD fluorescence intensity in absence of Cy5 acceptors. As shown in Figure 2B, the FRET efficiency increases with the increasing Cy5-labeled substrateto-QD ratio from 1/1 to 60/1; interestingly, Cy5 fluorescence intensity increases with the increasing Cy5-labeled substrate-toQD ratio from 1/1 to 48/1, but beyond the ratio of 48/1, Cy5 fluorescence intensity decreases. It is worth noting that the FRET efficiency and the Cy5 counts reach a plateau beyond the Cy5-labeled substrate-to-QD of 48/1 in the single-QD-based nanosensor (Figure 2A). In contrast, the FRET efficiency keeps increasing, and Cy5 fluorescence intensity decreases beyond the ratio of 48/1 in the bulk measurement (Figure 2B). The above deviations might be induced by the high local concentration of Cy5 in the bulk measurement, which might cause (1) the continuous quenching of QD donor by the excess Cy5 and (2) the inner-filter effect.61 In the bulk measurement, as long as Cy5s are within the efficient distance of FRET, they can quench the QDs no matter whether they are assembled on the surface of the QDs or not, resulting in the increase of calculated FRET efficiency with the increasing Cy5-labeled substrate-to-QD ratio even beyond the ratio of 48/1. The inner-filter effect can lead to the decrease of

shows the variance of Cy5 counts as a function of reaction time in the presence of 5 nM renin. With the cleavage of peptide substrates by renin, Cy5 acceptors are separated from the QD donor, resulting in the decrease of FRET efficiency. Consequently, Cy5 counts decrease with the reaction time. But Cy5 counts display no further decrease beyond 120 min, which might be attributed to either the complete losing of renin activity or the consumption of all available substrates by renin. In contrast, Cy5 counts remain unchanged during the reaction time of up to 180 min in the absence of renin. This result indicates that this single-QD-based nanosensor is specific to renin. Renin Proteolytic Assay. To accurately evaluate the enzymatic activity, we used the calibration curve in Figure 2A to obtain the concentration of noncleaved substrate. The concentration of cleaved substrate can be deduced from the difference in the concentration of noncleaved substrate between before and after the reaction. The proteolytic velocity is defined as the cleaved peptide substrate (nanomolar) per min. The initial velocity was measured in the presence of 0.5 nM renin and various substrate concentrations in 20 min of reaction at 37 °C to make sure that ∼80% of the substrates were unconsumed (i.e., in the initial-rate regime). The corresponding Michaelis 8849

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constants KM and maximal velocities Vmax were estimated using the Michaelis−Menten expression for excess substrate.62 V=

Vmax[S] d[P] = dt KM + [S]

(5)

where [P] is the concentration of reaction product, i.e., the cleaved peptide, [S] is the substrate concentration, and t is the reaction time. Figure 4 shows the analysis of Michaelis−

Figure 5. Variance of the reduction of Cy5 counts as a function of renin concentration. Inset: the reduction of Cy5 counts is a linear correlation with renin concentration in the range from 0 to 400 pM. Error bars show the standard deviation of three experiments. The substrate concentration is 4.8 μM, and the QD concentration is 0.1 μM in the reaction solution.

low as 25 pM is achieved. Notably, the sensitivity of the singleQD-based nanosensor has improved by more than 40-fold as compared with the method using EDANS/DABCYL as the fluorophore/quencher pair10 and more than 2-fold as compared with the method using 7-methoxycoumarin/DNP as the fluorophore/quencher pair.10 The improved sensitivity might be attributed to (1) the high FRET efficiency between the QD donor and Cy5 acceptor as a result of the capability of a single QD to couple multiple acceptors,21 and (2) the high signal-tonoise ratio of single-molecule detection.64 In addition, this single-QD-based nanosensor is straightforward without the involvement of any pretreatment and separation steps and extremely low cost with the consumption of minimum enzyme and the QDs.

Figure 4. Analysis of Michaelis−Menten kinetic parameters by the initial-rate method. The renin concentration is 0.5 nM, and the QD concentration is 0.1 μM in the reaction solution. Error bars show the standard deviation of three experiments.

Menten kinetic parameters by the initial-rate method. The experimental data were fit to eq 5 to obtain the kinetic parameters of KM = 5.9 ± 0.9 μM, Vmax = 49.8 ± 3.7 nM min−1, Kcat = Vmax/[enzymetotal] = 1.7 s−1, and Kcat/KM = 290 000 M−1 s−1. These results are in agreement with the previously reported values of KM = 5.5 ± 0.7 μM, Kcat = 1.9 s−1, and Kcat/KM = 350 000 M−1 s−1 for same enzyme obtained by the bulk fluorometric assay,10 suggesting that this single-QD-based nanosensor can be used to accurately evaluate the Michaelis− Menten kinetic parameters. In addition, it should be noted that a previous research demonstrated the deviations from classic Michaelis−Menten behavior at a QD interface due to the manifestation of nonMichaelis−Menten activity such as the loss of diffusion limitations.63 In our research, the QDs were added into the solution to form the QD/substrate/Cy5 complexes after the termination of substrate−renin reaction, thus efficiently circumventing the QD-induced loss of diffusion limitations and preventing the deviations from classic Michaelis−Menten behavior. Sensitivity of the Renin Assay. To investigate the sensitivity of renin assay, renin with different concentrations was measured by the single-QD-based nanosensor. In the presence of renin, the substrates are cleaved, leading to the decrease of FRET efficiency. Consequently, Cy5 counts decrease with the increasing renin concentration. As shown in Figure 5, the reduction of Cy5 counts increase with the increasing renin concentration in the range from 0 to 800 pM but reach a plateau beyond 800 pM. Notably, a linear correlation is obtained between the reduction of Cy5 counts and the renin concentration in the range from 0 to 400 pM (inset of Figure 5). The correlation equation is Nreduction = 33.6 + 0.9141C with a correlation coefficient of 0.9855, where Nreduction is the reduction of Cy5 counts and C is the renin concentration (picomolar), respectively. A detection limit of as

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CONCLUSION In summary, we have developed a single-QD-based nanosensor for sensitive detection of renin activity. Through the measurement of Cy5 counts by TIRF microscopy, the renin activity can be quantitatively evaluated at the single-molecule level. This single-QD-based nanosensor can measure not only the renin concentration but also the enzymatic velocity and the Michaelis−Menten kinetic parameters. In comparison with indirect radioimmunoassay,12,13 monoclonal antibody-based radioimmunometric assay,14 and organic dye- and proteinbased fluorescent methods,10,15−19 this single-QD-based nanosensor has significant advantages of simplicity, safety, low sample consumption, and high sensitivity. These advantages justify its potential for further applications in clinical diagnosis. In addition, this single-QD-based nanosensor can be extended to monitor a variety of important enzymatic biomarkers such as kinases and endonuleases.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86 755 86392211. Fax: +86 755 86392299. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 8850

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program 973 (Grant Nos. 2011CB933600 and 2010CB732600), the National Natural Science Foundation of China (Grant Nos. 21075129 and 11004213), the Guangdong Innovation Research Team Fund for Low-Cost Healthcare Technologies, the Natural Science Foundation of Shenzhen City (Grant No. JC201005270327A), the Fund for Shenzhen Engineering Laboratory of Single-Molecule Detection and Instrument Development, and the Award for the Hundred Talent Program of the Chinese Academy of Science.

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dx.doi.org/10.1021/ac302284s | Anal. Chem. 2012, 84, 8846−8852

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(64) Wazawa, T.; Ueda, M. In Microscopy Techniques; Rietdorf, J., Ed.; Springer-Verlag: Berlin, Germany, 2005; Vol. 95, pp 77−106.

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