FEBS Letters 588 (2014) 3553–3563

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Review

Single-enzyme kinetics with fluorogenic substrates:lessons learnt and future directions

http://dx.doi.org/10.1016/j.febslet.2014.06.0210014-5793/� 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

⇑ Corresponding authors.E-mail addresses: a.rowan@science.ru.nl (A.E. Rowan), k.blank@science.ru.nl

(K. Blank).

Petri Turunen, Alan E. Rowan ⇑, Kerstin Blank ⇑Radboud University Nijmegen, Institute for Molecules and Materials, Department of Molecular Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 May 2014Revised 4 June 2014Accepted 5 June 2014Available online 12 June 2014

Edited by Elias M. Puchner, Bo Huang,Hermann E. Gaub and Wilhelm Just

Keywords:Single-molecule fluorescenceEnzyme kineticsEnzyme dynamicsFluorogenic substrateNanophotonic structureZero mode waveguide

Single-molecule fluorescence techniques have developed into powerful tools for studying the kinet-ics of biological reactions at the single-molecule level. Using fluorogenic substrates, enzymatic reac-tions can be observed in real-time with single-turnover resolution. The turnover sequence containsall kinetic information, giving access to reaction substeps and dynamic processes such as fluctua-tions in the reaction rate. Despite their clearly proven potential, the accuracy of current measure-ments is limited by the availability of substrates with 1:1 stoichiometry and the signal-to-noiseratio of the measurement. In this review we summarize the state-of-the-art and discuss these lim-itations using experiments performed with a-chymotrypsin as an example. We are further provid-ing an overview of recent efforts aimed at the improvement of fluorogenic substrates and thedevelopment of new detection schemes. These detection schemes utilize nanophotonic structuressuch as zero mode waveguides or nanoantennas. Nanophotonic approaches reduce the size of theeffective detection volume and are a powerful strategy to increase the signal-to-noise ratio. Webelieve that a combination of improved substrates and novel detection schemes will pave the wayfor performing accurate single-enzyme experiments in biologically relevant conditions.� 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

Enzymes are dynamic molecules with the ability of catalyzingbiochemical reactions with high rate accelerations and specificity.The origin of this dynamic nature is a multidimensional energylandscape that does not only define the 3D structure but also thecatalytic function as well as regulation processes. Over the pastfew decades, single-molecule techniques, especially single-mole-cule fluorescence spectroscopy (SMFS) and microscopy, haveevolved into powerful tools for studying enzymatic reactions.Advances in measurement technology and the development offluorescence-based reporter systems [1–4] have improved the sen-sitivity and the time resolution to an extent such that dynamic pro-cesses during the catalytic reaction can be monitored in real-time,directly giving access to the sequence of events. Temporal fluctua-tions of enzyme behavior as well as heterogeneities between dif-ferent enzymes in the population can be determined directly insingle-molecule experiments. This information is inaccessible inensemble measurements.

Conformational changes can, for example, be monitored utiliz-ing Förster Resonance Energy Transfer (FRET). FRET is a sensitivespectroscopic ruler that reports on the distance between two fluo-rophores in close proximity. The energy transfer efficiency is adirect readout of the fluorophore distance and allows for detectingdistance changes in the range of several nanometers [5–12]. At thesingle-molecule level (smFRET), when the donor and acceptor fluo-rophores are attached to specific positions on an enzyme, theenergy transfer efficiency (EFRET) provides a direct readout ofconformational changes. Ha et al. [5] were the first to utilize thisprinciple for monitoring the conformational dynamics of staphylo-coccal nuclease. Fluctuations in EFRET occurred on the millisecondto second timescale, which is characteristic for functionally rele-vant conformational changes. The strength of this detectionscheme is at the same time one of its biggest weaknesses. In orderto obtain meaningful information about conformational motions,the locations of the donor and acceptor fluorophores need to beknown exactly. This requires prior knowledge of the enzyme struc-ture and an advanced specific labeling procedure [13–15].

smFRET can also be used for following reaction substeps andintermediate states of enzymes containing an optically activecofactor, such as in many oxidoreductases. The enzymatic turnovercycle can be observed directly when the cofactor cycles between anoxidized and a reduced state with different spectral properties

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[16–21]. A cofactor that absorbs light in only one of its oxidationstates can be used as a FRET acceptor. Labeling the enzyme witha donor fluorophore in close proximity to the cofactor will conse-quently lead to changes in the donor fluorescence intensity as afunction of the oxidation state of the cofactor. FRET labeling isnot required when the cofactor itself is fluorescent in its oxidizedstate [16–18]. In this case the redox cycling of the cofactor canbe followed directly.

One of the most intriguing discoveries of single-molecule enzy-mology has been the observation of temporal fluctuations in theactivity of enzymes, a phenomenon named dynamic disorder[16,22–25]. These temporal fluctuations in the rate-limiting stephave been the basis for the ‘fluctuating enzyme model’ that wasdeveloped to explain these observations [23]. This model relatesfluctuating rate constants with conformational changes, assumingthat each conformation has its own specific activity. A reliableidentification of dynamic disorder requires the detection of a largenumber of individual enzymatic turnovers. Sufficient statistics isrequired to accurately detect the underlying dynamic processesthat may span a large range of timescales. The acquisition of therequired data is only possible with a suitable reporter system thatallows for long measurement times. The reporter systems men-tioned above each possess certain technical limitations preventingsuch long measurement times and consequently the accurate anal-ysis of dynamic heterogeneities in the catalytic activity. smFRETdoes ‘only’ provide information about conformational changesbut does not directly detect enzymatic turnovers. Cofactor-baseddetection schemes are naturally limited to enzymes containingan optically active cofactor. For both detection schemes, the mea-surement is based on following the same fluorophore over the timecourse of the experiment. The measurement is consequently termi-nated by photobleaching of the fluorophore, limiting the dataacquisition time for one single enzyme to only a few seconds.

Fluorogenic substrates, which are converted by the enzyme intofluorescent product molecules, are in principle the only reportersystems that permit sufficiently long measurements. Fluorogenicsubstrates are the ideal system for studying enzyme kinetics as a

Fig. 1. Single-turnover detection with fluorogenic substrates. (a) The laser of a confocalslip. Every fluorophore produced by the enzyme is recorded in real-time with single-phoThreshold analysis (b) or change point analysis (c) are used to assign the ON- and OFF-

new fluorophore is produced during every enzymatic turnover[22–24,26–31]. In this paper, we review the current state of sin-gle-enzyme experiments focusing on fluorogenic substrates asreporter systems. We will first describe the technological require-ments and the basic experimental setup used for performing theseexperiments. In the following, we will describe a recent single-molecule study of a-chymotrypsin as an example to highlight theunique information that can be obtained in such an experiment.Using this same example, the current limitations of the substratedesign and the measurement setup will be discussed together withrecent developments aimed at overcoming these limitations. Theseinclude improved fluorogenic substrates as well as single-moleculedetection schemes. These technological advances have the poten-tial to improve the signal-to-noise ratio of the measurement andwill ultimately help to perform single-enzyme experiments underbiologically relevant conditions while at the same time allowingfor a more accurate kinetic analysis.

2. Single-turnover detection

The ideal single-turnover measurement requires the directobservation of every enzymatic turnover with a signal-to-noiseratio (SNR) high enough for accurate data interpretation (Fig. 1a).Fluorogenic substrates provide this direct readout of individualenzymatic turnovers. They consist of a fluorophore linked to afunctional group that is recognized by the enzyme. This functionalgroup alters the photochemistry of the fluorophore, making it non-emissive. After enzymatic cleavage of the functional group, thefluorophore recovers its fluorescence. To be able to follow thesequence of turnovers, the enzyme needs to be immobilized onthe surface of a glass cover slip. The enzyme itself is also fluores-cently labeled so that every individual enzyme can be located onthe surface. Upon the addition of substrate, the fluorescence signalat the position of one enzyme is recorded. Enzyme activity resultsin the formation of fluorescent product molecules at this specificposition on the surface. Using a confocal microscope, every productmolecule is detected as a fluorescence burst above the background

microscope is focused onto the position of an immobilized enzyme on a glass coverton sensitivity yielding the arrival time (‘‘macro-time’’) of every individual photon.

times to the photon arrival time trace.

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noise. The product fluorophore is detectable only for a short timewindow after it has been created by the enzyme. It quickly exitsthe detection volume by diffusion. As every turnover yields anew fluorophore, the measurement time is not limited by photo-bleaching of the fluorophore but by the accumulation of fluores-cent product molecules, which results in a reduced SNR.

The choice of fluorophore is critical since its brightness directlydetermines the SNR. The measurement setup does not only need tobe sensitive enough to detect every single product molecule, butalso needs to have a high temporal resolution. These criteria arefulfilled by a fluorescence microscope with confocal optics andavalanche photodiode (APD) detectors [1–4]. In a confocal micro-scope that is optimized for single-turnover measurements, a colli-mated laser beam is focused into a diffraction-limited spot using amicroscope objective with a high magnification and a high numer-ical aperture. In the most commonly used epi-fluorescence config-uration, fluorescence emission from the sample is collected withthe same objective, spectrally separated from the excitation lightwith optical filters and guided to the detector(s). Before reachingthe detector, the emitted light is focused through a small pinhole.Consequently, only fluorescence originating from the small focalvolume is collected, reducing the size of the detection volume toapproximately 1 femtoliter. This small detection volume facilitatesthe required SNR for detecting individual fluorophores. The APDdetector provides single photon sensitivity and the required timeresolution.

Using the setup described above, only the absolute photon arri-val time (‘‘macro-time’’) is recorded, which gives the fluorescenceintensity (photons/time) of every individual product fluorophore.Extra information about the catalytic process can be obtainedwhen the fluorescence lifetime is determined simultaneously in aso-called time-resolved measurement [30,32,33]. For these mea-surements the generated fluorophore is excited periodically witha picosecond-pulsed laser. The emitted photons are detected withtime-correlated single-photon counting (TCSPC) [34,35]. Whenusing TCSPC, the arrival time relative to the last laser pulse (the‘‘micro-time’’) is recorded for each photon. Once a sufficientnumber of micro-times have been collected, the correspondingmicro-time histogram allows for determining the lifetime of thefluorophore. The time resolution of modern, fast APDs and photoncounting electronics is in the picosecond range, yielding lifetimeswith high accuracy.

The data analysis procedure for extracting kinetic informationstarts with the recorded photon arrival time trace (macro-timetrace). The first and most important goal is to obtain a binaryON–OFF trace (ON–OFF assignment). This binary trace is the basisfor the kinetic analysis of the system. In this binary trace, the timeintervals characterized by high (ON) photon count rates representthe enzymatic turnovers, while the low-intensity intervals repre-sent the background noise (OFF). The Poisson nature of single-pho-ton detection and the background noise consisting of productdiffusion and inelastic scattering add challenges to the ON–OFFassignment. Two principally different approaches are availablefor the ON–OFF assignment. The most commonly used approachis the so-called threshold method (Fig 1b). When using thismethod, the photon arrival time trace is first binned to obtain aphoton count (intensity) time trace. A threshold is then appliedto the photon count time trace to separate the ON- and OFF-levels.An alternative and more recently developed method is changepoint analysis [29,36–38]. Change point analysis is a statisticalmethod for identifying changes in the photon count rate directlyin the photon arrival time trace (Fig 1c). It does not require bin-ning. Instead, the photon statistics before and after each photonis compared using a statistical hypothesis test. This test is per-formed along the complete time trace and evaluates the log-likeli-hood ratio of the probability for each photon to be an intensity

change point. The photon with the highest probability is assignedto be a change point. The time trace is then cut into two fragmentsat the change point photon and the algorithm is used for bothfragments of the time trace. The process is repeated recursivelyuntil no intensity change points are found in the time traceanymore.

Once the ON–OFF binary trace has been obtained, multiplestrategies are possible to extract the desired kinetic information[3,23,29,39–41]. The kinetic constants can be determined by fittingthe probability distribution function of the OFF-time histogramwith a model describing the kinetic process. In the case of a reac-tion with a single rate constant the OFF-time histogram follows amono-exponential decay (straight line in a log-linear plot). In thecase of a dynamic enzyme with multiple active states, the contri-butions from multiple reaction rates result in a multiexponentialdecay profile. For most experimental data obtained so far OFF-timehistograms with a concave shape have been obtained [16,22–25,27]. It was found that these histograms were fitted best witha stretched exponential function. This ‘‘stretched exponential’’was used to describe an enzymatic reaction that exhibits a distri-bution of rate constants (dynamic disorder). Instead of using theOFF-time distribution, also the autocorrelation function of theOFF-times can be used to extract information about the underlyingkinetics. More complex kinetic behavior, such as dynamic disorder,leads to correlations between consecutive OFF-times, indicatingthat OFF-times of similar length are more likely to directly followeach other. The difficulty in obtaining and interpreting the kineticinformation is discussed in the example below.

3. Single-molecule study of a-chymotrypsin

a-Chymotrypsin is a well-studied digestive protease. Catalyzingthe hydrolysis of peptide bonds, its main function is the break-down of proteins into amino acids. Its activity is tightly regulatedand highly pH-dependent [42,43]. The enzyme exists in a confor-mational equilibrium involving an active and an inactive confor-mation [43]. The related conformational changes occur on a timescale from milliseconds to seconds allowing the conformationaldynamics to be detected in single-molecule experiments. Adetailed single-molecule study of a-chymotrypsin has beendescribed in a series of papers [29,30,44]. The goal of this studywas to systematically investigate the performance of the currentlyused techniques described above and to improve the accuracy ofthese methods.

One key feature of many commonly used fluorogenic substratesis the presence of two functional groups. These double-substitutedfluorogenic substrates require two enzymatic cleavage steps beforethe fully fluorescent product is obtained. In most cases the inter-mediate shows fluorescence in the same wavelength range, how-ever, its brightness is lower. This significantly complicates itsdetection in a single-enzyme measurement. Turnovers might bemissed preventing an accurate kinetic analysis. For the a-chymo-trypsin experiments described here the fluorogenic substrate wasbased on the commonly used fluorophore Rhodamine 110(Rh110). The substrate carries two short peptides coupled to thetwo amine groups of the fluorophore to yield the fluorogenic sub-strate (succinyl-AlaAlaProPhe)2-Rhodamine 110 (sAAPF2-Rh110).The peptide sequence was chosen to match the amino acid prefer-ence of a-chymotrypsin. In the case of Rh110, the intermediate isnot only bright enough to be detected [30] but also has a differentfluorescence lifetime than the product (Fig. 2) [30,44].

The activity of single a-chymotrypsin molecules, immobilizedin an agarose gel, was measured using a confocal detection schemeequipped with TCSPC. After performing the ON–OFF assignmentwith change point analysis, the fluorescence lifetimes were deter-mined for each individual ON- and OFF-state. This lifetime analysis

Fig. 2. Fluorescent reporter system for measuring the activity of a-chymotrypsin at the single-molecule level. The substrate is based on the fluorophore Rhodamine 110. Itcarries two peptides bonds that can be cleaved by a-chymotrypsin. Both the final product and the mono-substituted reaction intermediate are fluorescent. Differences in thequantum yields U and the fluorescence lifetimes s allow for a discrimination of these two fluorescent molecules.

Fig. 3. Comparison of the threshold method with change point analysis. Shown areexperimental data obtained for an a-chymotrypsin catalyzed reaction. (a) Depend-ing on the data analysis method used, the OFF-time histograms show a differentshape. (b) When using the threshold method, correlations between subsequentturnovers are observed in both the autocorrelation plot and in the 2D-correlograms(n = 1; timescale 40 ms). No correlations are detected when using change pointanalysis. Reprinted with permission from Terentyeva et al. (2012) ACS Nano 6,346–354 [29]. Copyright 2012 American Chemical Society.

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revealed that only the reaction intermediate was produced in theenzymatic reaction as only the corresponding lifetime of 2.7 nswas found for the ON-states. Also the OFF-states showed an aver-age lifetime of 2.5 ns. This result suggests that the backgroundphotons mostly originate from intermediate molecules diffusingthrough the detection volume. The measurement was performedat a substrate concentration of 30 lM, a factor 4x higher than theapparent KM value of the two-step reaction. Under these conditionsthe substrate concentration is far higher than the intermediateconcentration in a single-enzyme experiment. As both substrateand intermediate compete for binding to the enzyme, the hydroly-sis of the intermediate is very unlikely. Formation of the Rh110product would only be possible if the intermediate would be con-verted to product directly after it has been formed (intermediatechanneling) [45]. The results do not show any evidence of channel-ing, enabling a direct insight into the reaction mechanism of thestudied two-step reaction. This result allowed the system to betreated as a reaction with 1:1 stoichiometry, which is ultimatelyrequired for studying the kinetics of the conformational changeand the possible presence of dynamic disorder.

The ability of detecting the intermediate clearly simplifies thekinetic analysis. At the same time, however, it complicates theON–OFF assignment, which precedes the kinetic analysis. Theproblem is the low brightness of the intermediate (U = 0.31) thatcauses a low SNR. In the case of the described a-chymotrypsinexperiments the SNR was only �2.5. At SNRs below 5 the intensitydistributions of the ON- and OFF-states overlap, hampering anaccurate ON–OFF assignment. For such low SNR cases, the accuracyof the commonly used binning and thresholding method has beensystematically compared with change point analysis [29]. Bothmethods were tested using large sets of simulated data with differ-ent SNR and intensity levels. Binning and thresholding generallyoverestimates the number of short OFF-times. In contrast, thenumber of short OFF-times is underestimated when using changepoint analysis. This problem is especially dominant when the pho-ton count rate is low ( 5). When the SNR was low(SNR < 3), both methods failed to reproduce the predicted OFF-time histograms accurately.

For the experimental data with a SNR of 2.5 the different meth-ods gave different turnover rates (26.3 s�1 with the thresholdmethod and 14.5 s�1 with change point analysis). In addition, theyproduced significantly different OFF-time histograms and autocor-relation results (Fig. 3). The largest difference in the OFF-histo-grams is observed in the short OFF-time region where thresholdanalysis produces a larger number of short OFF-states (Fig. 3a).This over-representation of short OFF-times resulted in the typi-cally observed concave shape of the OFF-histogram that was fitted

with a stretched exponential in previous research. The over-repre-sentation of short OFF-times also explains the higher turnover ratedetermined with the threshold method.

More interesting is the appearance of correlations between con-secutive short OFF-times in the 2D-correlogram when the thresh-old method is used (Fig. 3b). These correlations are absent in thecase of change point analysis. These results indicate that the obser-vation of these correlations and the over-representation of shortOFF-times are artifacts originating from threshold analysis. Thefact that change point analysis does neither yield correlationsbetween consecutive events nor stretched exponential OFF-timehistograms questions the interpretation of previous single-enzymeexperiments where similar results were obtained [16,22–25]. Thissystematic analysis clearly highlights that only data with a highSNR will allow a more detailed kinetic analysis of a-chymotrypsin

P. Turunen et al. / FEBS Letters 588 (2014) 3553–3563 3557

and other enzymatic reactions in general. Only then can any con-clusions be drawn about the presence or absence of dynamicdisorder.

In summary, the single-molecule experiments performed witha-chymotrypsin illustrate the unique information that can beobtained with the current measurement technology. Novel insightinto the order of reaction substeps has been obtained: no productmolecules are formed by intermediate channeling when using dou-ble-substituted substrates. The low SNR resulting from the lowbrightness of the intermediate is the main bottleneck that limitsthe information that can be extracted from these measurements.Advanced data analysis methods alone cannot solve this problem.Even change point analysis, as an objective method, cannot detectthe intensity change points reliably enough if the SNR of the data isnot sufficiently high. The absence of dynamic disorder in the a-chymotrypsin measurements does not exclude its existence. Newdevelopments addressing both the fluorogenic substrate reportersystems and the measurement technology are required beforesingle-turnover measurements can facilitate a more detailedkinetic analysis and ultimately become a standardized tool forbiochemistry.

Fluorogenic substrates combining 1:1 stoichiometry with ahigher brightness are obviously needed. Despite the high time res-olution and single-molecule detection efficiency, confocal micros-copy has its limitations when considering the experimentalconditions and the biological relevance of single-enzyme measure-ments. a-chymotrypsin is a unique example allowing single-turn-over detection at substrate concentrations above the (apparent) KMvalue of the reaction. This is not possible for a large number ofother enzymes. Single-molecule occupancy in a diffraction-limited,femtoliter-sized detection volume corresponds to picomolar tonanomolar fluorophore concentrations. This often prevents theuse of substrate concentration above the KM value. The use ofnanophotonic structures is one strategy that has the potential toovercome these limitations. These structures facilitate a drasticreduction of the size of the detection volume by either confiningor locally enhancing the excitation light. In the following sectionswe will review recent efforts aimed at the development of next-generation fluorogenic substrates and of detection schemes basedon nanophotonic structures. When combined, these developments

Fig. 4. Examples of Rhodamine 110-based substrates with 1:1 stoichiometry. (a) Urederivative with a high brightness (e.g. morpholinecarbonyl Rhodamine 110; MC-Rh11fluorescent product molecule. The second amino group can be used for coupling the peSingapore Green (SG) contains only one amino group. Also in this case, only one functRhodamine 110 (HMRG) only one peptide needs to be coupled to obtain a non-fluoresc

will ultimately lead to single-enzyme measurements with drasti-cally improved SNRs under biologically relevant conditions.

4. Next-generation fluorogenic substrates

Fluorogenic substrates are very powerful reporter systems. Theexperiments described above clearly show their potential for mon-itoring long reaction sequences. They also highlight one of the keyproblems of current substrate designs. Rhodamine 110 (Rh110)and fluorescein are commonly used for the synthesis of fluorogenicsubstrates. They are both bright fluorophores that are easilydetected in a single-molecule experiment. But they contain twofunctional groups that can be substituted during substrate synthe-sis. Mono-substituted derivatives of Rh110 are fluorescent andbright enough to be detected in a single-turnover experiment,however, with a much lower SNR [30,46]. In contrast, mono-substituted fluorescein derivatives appear to have a very low quan-tum yield in the range from 0.0003 to 0.22 [47–50]. Even though nodata is currently available, it appears highly unlikely that mono-substituted fluorescein derivatives can be detected at the single-molecule level.

For both Rh110 and fluorescein, next-generation substrateswith only one enzyme-cleavable group have been developed inrecent years. The first step towards improved Rh110-based sub-strates with 1:1 stoichiometry was the development of Rh110substituted with one urea-group, such as morpholinecarbonyl-Rhodamine 110 (MC-Rh110; Fig. 4a) [44,51]. The brightness ofmono-substituted Rh110 derivatives depends on the electronega-tivity of the substituent [44]. Substituents with a higher electro-negativity cause a stronger distortion of the symmetry of thexanthene unit of the fluorophore, reducing both its extinction coef-ficient and its quantum yield. When comparing MC-Rh110 with amono-functionalized peptide-Rh110, MC-Rh110 shows a signifi-cantly higher extinction coefficient (e = 52 000 M�1 cm�1) andquantum yield (U = 0.60) than the peptide-derivative(e = 24 600 M�1 cm�1; U = 0.31; Fig. 2). As the urea-bond is notcleaved enzymatically, the relatively bright MC-Rh110 can be usedas a new fluorophore. The remaining amino group of MC-Rh110can be coupled to a peptide to yield a fluorogenic substrate with1:1 stoichiometry (Fig. 4a).

a-substitution of one amino group of Rhodamine 110 yields a mono-substituted0). As the urea group is not cleaved enzymatically, MC-Rh110 can be used as a

ptide, yielding a substrate with 1:1 stoichiometry. (b) The asymmetric fluorophoreional group is available for coupling the peptide. (c) In the case of hydroxymethylent derivative due to spirocyclization of the structure.

Fig. 5. Distribution of KM values containing all enzyme-substrate pairs in theBRENDA database. Data taken from http://www.brenda-enzymes.info/.

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Another strategy to obtain a fluorophore with only one aminogroup for coupling the peptide is the direct replacement of theamino group with a different functional group. This strategy hasbeen followed when synthesizing the fluorophore Singapore Green(SG; Fig. 4b) [52]. SG is a hybrid of Rh110 and the fluorescein ana-logue Tokyo Green carrying one amino and one hydroxyl group atthe xanthene ring system. SG has a quantum yield of U = 0.5 andan extinction coefficient of e = 28500 M�1 cm�1. These values aresimilar to mono-substituted peptide-Rh110 derivatives so that sin-gle-molecule detection should be possible. A more recent approachis based on a mono-substituted hydroxymethyl derivative ofRh110 (HMRG; Fig. 4c) [53]. Coupling of only one peptide to HMRGinduces a spirocyclization reaction that disrupts the conjugatedelectron system of the xanthene unit. In this way a non-fluorescentcompound is obtained. After enzymatic cleavage of the peptide, thefluorescent form of HMRG (e = 57 000 M�1 cm�1; U = 0.81) isrecovered.

Besides yielding 1:1 stoichiometry, the Rh110 derivatives MC-Rh110 and SG have one additional advantage. Depending on theamino acid sequence of the peptide coupled, the substrate mightbecome less water-soluble, limiting the highest concentration thatcan be used in the experiment. In the case of MC-Rh110, the mor-pholinecarbonyl group can be readily replaced with a different andmore water-soluble urea-substituent. In the case of SG, the hydro-xyl group can, for example, be functionalized with a short, water-soluble poly(ethylene glycol) chain. Overall, asymmetric fluoro-phores provide numerous possibilities for introducing additionalfunctionality into these fluorogenic substrates, improving theirproperties.

In parallel to the above efforts aimed at the development ofmono-substituted Rh110 derivatives, several mono-substitutedfluorescein derivatives have been synthesized. In analogy to MC-Rh110, one hydroxyl group of fluorescein can be converted into amethoxy group to yield the fluorophore 3-O-methyl fluorescein.This fluorescein derivative has been used to synthesize the phos-phatase substrate 3-O-methyl fluorescein phosphate [54]. To bestof our knowledge nothing is known about the brightness of thisfluorophore in comparison to other weakly fluorescent mono-substituted fluorescein derivatives [47–50]. A more controlledstrategy for obtaining fluorescein derivatives with 1:1 stoichiome-try is based on tuning the redox potential between the xantheneunit and the benzene ring [55]. This redox potential has a directinfluence on the photophysical properties of fluorescein deriva-tives. Making use of the fluorescein derivative Tokyo Green, itwas possible to tune the system in a way that the coupling of anenzyme-cleavable group changed the redox potential such thatthe quantum yield was lowered to U = 0.01. After enzymatic cleav-age the redox potential is shifted back to its original value and thefluorescence was recovered (U = 0.85). In this way, enzyme sub-strates for b-galactosidase [55] and alkaline phosphatase [56] havebeen designed.

None of the mono-substituted Rh110 and fluorescein deriva-tives described above have been tested in single-molecule experi-ments so far. Even though they appear to have a high potential, it iscurrently not clear if the substrates are indeed ‘dark’ and the prod-ucts sufficiently bright for single-turnover experiments. In order toobtain a high SNR, the residual fluorescence of the substrate iscrucial and critically determines the highest useable substrate con-centration. In currently used confocal detection schemes it is, forexample, impossible to use the mono-functionalized peptide-Rh110 intermediate as a substrate, even though this is possiblein ensemble experiments. In a single-molecule experiment, severalof these substrate molecules are always present in the detectionvolume. Even though the brightness of these molecules is low, theywould still generate a strong fluorescence background reducing theSNR. This problem can be overcome with the use of improved

detection schemes based on nanophotonic metal structures thatreduce the effective detection volume.

5. Nanophotonic structures for single-enzyme experiments

The maximum concentration that still allows single fluorophoreoccupancy in the detection volume of diffraction-limited optics isin the low nanomolar range. This is demonstrated routinely in fluo-rescence correlation spectroscopy (FCS) experiments where molec-ular diffusion can be measured with a high SNR [57,58]. Thesemaximally allowed concentrations could become a problem in sin-gle-enzyme experiments with fluorogenic substrates. Even themost pure substrates contain at least 0.1% of fluorescent productmolecules. This is a direct consequence of the synthetic procedureused to prepare these substrates, where the fluorophore is one ofthe starting materials. Following coupling of the functional groups,the obtained fluorogenic substrate needs to be separated from thestarting material (fluorophore). Even with the best purificationprocedure, trace amounts of fluorophore always remain in thenewly synthesized substrate. Autohydrolysis of the substrate canfurther lead to an increase in the fluorophore concentration duringthe experiments, thereby causing a decrease in the SNR over time.Whereas trace amounts of impurities are usually not a problem inensemble measurements, they have a significant effect on the SNRin single-molecule experiments. Assuming 0.1% of fluorescentimpurities, it is not possible to use substrate concentrations above10 lM as the substrate will contain 10 nM fluorescent impurities.As the average KM value of an enzyme is in the range between100 lM and 1 mM (Fig. 5), single-turnover measurements are gen-erally limited to substrate concentrations below the KM value. Inthis range, substrate diffusion to the active site might becomethe rate-limiting step and determine the kinetic behavior of theenzyme.

Reducing the size of the effective detection volume overcomesthese problems, but is a technologically challenging solution. Manytechnological developments during the past decade have facili-tated a reduction of the effective size of the detection volume[59]. When using Total Internal Reflection (TIR) illumination thesize of the excited volume is defined by an evanescent wave thatdecays exponentially from the sample surface. In this way theexcited volume is reduced in the z-dimension. As a result, theeffective detection volume is approximately 2.5-fold smaller thanfor confocal detection schemes [60,61]. The Near-Field ScanningOptical Microscope (NSOM) also makes use of an evanescent waveto excite fluorescence on a sample surface. [62,63]. The excited vol-ume is spatially confined at a scanning probe tip that contains anaperture with a typical diameter of 50 nm. As the light intensityis localized at the aperture, the size of the detection volume isreduced by approximately 1000-fold. NSOM is mainly an imagingtechnique that is not easily applicable in single-turnover

http://www.brenda-enzymes.info/

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experiments. Tip fabrication is complex and the tip would have tobe placed close to the enzyme for the whole duration of themeasurement.

5.1. Zero mode waveguides

As demonstrated by the NSOM, nanophotonic structures canoffer a powerful strategy for confining the excitation light. Zeromode waveguides (ZMWs) use the same fundamental operatingprinciple as NSOM, but are more easily implemented for single-turnover experiments. ZMWs are nanophotonic structures thatconsist of an array of mostly round holes milled in an opaquemetallic film supported by a glass surface (Fig. 6). When the holediameter is well below the excitation wavelength, no light abovea certain cut-off wavelength can propagate through the hole thuscreating a decaying evanescent wave at the entrance of this aper-ture. This cut-off wavelength and the attenuation factor of thepropagating modes depend on the shape and size of the aperture[64–66]. In theory, the excited volume in the apertures of a ZMWarray is defined by the diameter of the aperture and the character-istic decay length of the evanescent wave. ZMW arrays are usuallyfabricated using electron-beam lithography or focused ion-beammilling. First, a 150-400 nm thick metal film is deposited on amicroscope cover slip. Aluminum or gold is generally used as amaterial. In the next step, apertures with a typical diameter rang-ing from 30 to 250 nm are milled into the metal. The size of theseapertures ensures cut-off for wavelengths in the 450-700 nmrange, which is the wavelength range required for the most com-monly used organic fluorophores.

The potential of ZMW arrays for reducing the size of the detec-tion volume was first demonstrated in an FCS measurement wherethe diffusion of Rh110-labeled dCTP nucleotides was observed. Inthis experiment apertures with a diameter of 43 nm were fabri-cated in an aluminum film. For this aperture size the effectivedetection volume was estimated to be approximately 20 zL, whichis three orders of magnitude smaller than with diffraction-limited

Fig. 6. Typical layout of a single-turnover experiment in a zero mode waveguide(ZMW) array. The ZMW array is fabricated in a gold or aluminum layer deposited ona glass cover slip. Individual enzyme molecules are immobilized on the glasssurface at the bottom of the holes where the intensity of the evanescent field ishighest.

detection schemes [64]. In the FCS experiment, this small size ofthe detection volume facilitated single-molecule occupancy at afluorophore concentration of 10 lM. The FCS measurement furthershowed a significant reduction of the diffusion time of Rh110-dCTPto only a few microseconds. This is one order of magnitude fasterthan in a diffraction-limited detection volume and further confirmsthe reduced size of the detection volume.

The major advantage of using ZMWs instead of NSOM for sin-gle-turnover experiments is that they act as a nanoaperture anda reaction vessel at the same time (Fig. 6). Enzymes can be immo-bilized on the glass surface at the bottom of the nanostructurewhere the confined excitation intensity is highest. This approachhas been used for following the activity of T7 DNA polymerase atthe single-enzyme level [64]. The enzyme was immobilized in43 nm-sized apertures in an aluminum waveguide structure. Theenzyme concentration was adjusted such that statistically only afraction of apertures contained a single enzyme. Polymerase activ-ity was then measured using the intrinsically fluorescent substratecoumarin-dCTP at a concentration of 7.5 lM. Fluorescence burstswere detected and could be clearly distinguished from the back-ground caused by the diffusion of fluorescent nucleotides. Enzy-matic turnovers could further be discriminated from diffusionevents based on the residence time of the fluorophore in the detec-tion volume, which was significantly longer in the case of a nucle-otide incorporation event. The ability to use an intrinsicallyfluorescent substrate based on a coumarin dye highlights theunique potential of nanostructures for single-enzyme experiments.Coumarin derivatives have a low excitation coefficient, which isnot sufficient for single-molecule measurements in diffraction-lim-ited volumes [67]. In the ZMW structure, however, the significantlysmaller detection volume facilitates the discrimination of this fluo-rophore from the fluorescent background.

Following these proof-of-principle experiments, the measure-ment setup was developed further to facilitate DNA sequencing[68]. To be able to monitor nucleotide incorporation in real-time,the four different nucleotides were labeled with spectrally distin-guishable fluorophores. In this way, nucleotide incorporation couldbe monitored for thousands of single polymerase molecules simul-taneously using a large ZMW array and an advanced parallel con-focal multichannel detection scheme [69]. Each nucleotideincorporation event was detected as a fluorescent burst in one ofthe spectral channels with a high SNR (SNR = 25) allowing for thespecific assignment of the different DNA bases. In addition toobtaining the nucleotide sequence, the data also contained kineticinformation, i.e. the duration of each nucleotide incorporationevent. This information has, for example, been used for extractingdifferences in the polymerase reaction rate for different methyla-tion states [70].

Despite their high potential, ZMWs are still only rarely used forstudying single-enzyme kinetics. One important drawback, limit-ing the attractiveness of the ZMW-based detection system, is thestochastic nature of the enzyme immobilization procedure. Con-sidering the theoretical Poisson limit, maximally 37% of the aper-tures can be ‘filled’ with exactly one single enzyme when addingan enzyme solution to the nanostructure. Moreover, the immobi-lized enzymes cannot be specifically placed in the center of theaperture. Individual enzyme molecules are located at different dis-tances from the metal wall of the aperture resulting in heterogene-ities in the fluorescence excitation and emission properties. Toovercome these problems, an atomic force microscope (AFM)based ‘‘cut-and-paste’’ technique was recently developed [71,72].With this technique an individual molecule can be picked up withthe AFM cantilever, transported and subsequently placed into aZMW aperture. The cut-and-paste process can be repeated untilall apertures are filled with exactly one molecule. Resulting fromthe high positional accuracy of the AFM, this method also ensures

3560 P. Turunen et al. / FEBS Letters 588 (2014) 3553–3563

that every molecule is always placed in the center of the apertureso that heterogeneities in the fluorescence properties are reduced.In a very recent report, DNA origami was introduced as an alterna-tive strategy to improve the process of placing single moleculesinto the apertures [73]. With DNA origami [74,75] well-definedDNA nanostructures can be assembled that allow for the position-ing of fluorophores, nanoparticles and biomolecules. On a DNA ori-gami scaffold the position of each oligonucleotide is known withnanometer precision. Oligonucleotides modified with functionalgroups can therefore be integrated into the origami structure atpredefined positions. Using this approach, disc-shaped nanoadap-ters were designed that matched the size of the apertures. Thenanoadapters were immobilized on the surface of the ZMW via abiotin-neutravidin interaction, thereby ensuring that maximallyone nanoadapter was present per aperture. The nanoadapters werefurther labeled with a single ATTO647N dye to allow for theirdetection. In this way, single-molecule occupancy was observedin 60% of the apertures, thereby overcoming the theoretical Poissonlimit of 37%.

5.2. Fluorescence enhancement by metal nanostructures

One characteristic feature of ZMWs is that they do not only con-fine the excitation volume. The nanostructure can also enhance thesignal detected from a fluorophore thereby improving the SNReven further [76–78]. An enhancement of 6.5-fold was, for exam-ple, reported for the fluorophore Rhodamine 6G in the 150 nmholes of an aluminum ZMW [76]. A 12-fold enhancement wasfound for Alexa Fluor 647 in a gold structure with 120 nm holes[77,78]. In these examples, the enhancement originates from theenergetic coupling of the fluorophore with electron oscillations(surface plasmons) on the metal surface. If these surface plasmonsare resonant with the excitation or emission wavelength of thefluorophore, they can enhance the fluorescence [65,79].

The presence of surface plasmons affects both fluorescence exci-tation and emission. The plasmons can increase the excitationintensity by locally enhancing the optical field inside the aperture[76]. The surface plasmons further influence the de-excitation path-ways causing an increased quantum yield and a shorter fluores-cence lifetime [78]. The enhancement is stronger for lowquantum yield fluorophores. It allows the detection of these fluoro-phores in a ZMW structure with a SNR improved by one order ofmagnitude [80], eventually facilitating their use for fluorogenicsubstrates in single-enzyme experiments. The enhancement factordoes not only depend on the fluorophore but also on the metal andthe geometry of the nanostructure. Compared to aluminum, goldpossesses stronger surface plasmon resonances in the visible range.This directly leads to a stronger fluorescence enhancement in the600–700 nm range, but can also influence the fluorescence signalin an unfavorable way at lower wavelengths. The geometry of theaperture and the surrounding metal structure further influencethe emission directivity. Ideally using specifically designed nano-structures, the emitted photons can be directed towards the detec-tor [65,81]. A fluorescence enhancement of 120-fold combined witha 13.6-fold increase in emission directionality was recently demon-strated for the fluorophore Alexa Fluor 647 when the ZMW aperturewas surrounded by periodic corrugations [82]. These results showthat an optimization of the nanostructure parameters is a promis-ing route towards increasing the enhancement factor and thepotential of ZMW structures in general.

5.3. Optical nanoantennas

ZMWs make use of nanoapertures for reducing the effectivedetection volume and for enhancing the local excitation field. Thiscan also be obtained with nano-sized optical antennas that are

specifically designed to harness plasmonic effects. In contrast toZMWs, the antenna geometry is designed such that it optimizesthe local excitation field. It concentrates the excitation intensityfrom the surroundings and creates a localized excitation hotspot[83,84]. A metal nanoparticle can be considered as the simplestoptical nanoantenna. A 10-fold fluorescence enhancement has,for example, been reported for the fluorophore Nile Blue in theimmediate vicinity of gold or silver nanoparticles [85]. Theenhancement factor is strongly dependent on the particle-fluoro-phore distance. At very short distances (

Fig. 7. Typical optical nanoantenna designs. (a) In the case of a bowtie nanoantenna the excitation hotspot is located between the tips of the bowtie. The gap distance liesbetween 5 nm and 80 nm. (b) The ‘antenna-in-box’ design combines an antenna structure made from two half-spheres (gap distance between 12 nm and 40 nm) with arectangular nanoaperture. (c) Using a well-defined DNA origami structure, an antenna can be assembled on the DNA template using two nanoparticles (gap distance 23 nm).All nanostructures are made of gold.

P. Turunen et al. / FEBS Letters 588 (2014) 3553–3563 3561

alternative strategy to tackle the immobilization problem. Thisnanoscale assembly technique has been used to create a DNAnanopillar carrying two gold nanoparticles with a gap distance of23 nm (Fig. 7c) [87]. This DNA nanopillar was further functional-ized with several biotin molecules allowing its upright attachmentto a neutravidin functionalized microscope cover slip. Most impor-tantly, the nanopillar contained a special docking site for the mol-ecule of interest in the antenna hotspot between the nanoparticles.When one ATTO647N fluorophore was positioned in the hotspot, a117-fold fluorescence enhancement was observed. Subsequently,the conformational dynamics of the Holliday junction were visual-ized with this setup. Unfortunately, no measurements with freelydiffusing fluorophores have been carried out yet, so that no infor-mation is available about the SNR that could be achieved in sin-gle-enzyme measurements.

In summary, nanophotonic structures have the potential toovercome current bottlenecks in single-enzyme experiments. Theconfinement of the effective detection volume allows the use ofhigher substrate concentrations while ensuring a high SNR. Thefluorescence enhancement effect further facilitates the use of sub-strates based on fluorophores with a low brightness. In combina-tion, this gives access to a larger number of substrate designswith 1:1 stoichiometry that can be used in the biologically relevantconcentration range. ZMW nanoapertures have already developedinto a powerful detection scheme for single-turnover detection.Optical nanoantennas, designed to optimize the local fieldenhancement, are a possible alternative approach. They are underintensive development and a number of designs have been pro-posed in the past few years. Initial results with biological systemsare very promising and their implementation for single-enzymeexperiments can be expected in the near future.

6. Conclusions

Single-enzyme experiments allow for studying the kinetics ofenzymatic reactions in real-time, giving access to possible tempo-ral heterogeneities in the reaction rate. Fluorogenic substrates areideal reporter systems. They allow for following the catalytic reac-tion for extended periods of time thereby providing the timesequence (and statistics) of a large number of individual enzymaticturnovers. Current bottlenecks, such as the stoichiometry of thesubstrates and the low SNR of diffraction-limited detectionschemes, complicate the data analysis and limit the informationthat can be extracted from single-enzyme experiments. New devel-opments need to focus on strategies that facilitate the accuratedetection of every individual turnover with a high SNR in orderto facilitate a detailed kinetic analysis. The development of next-generation fluorogenic substrates with 1:1 stoichiometry and theimplementation of new detection schemes based on ZMW nanoap-ertures and optical nanoantennas are crucial improvements. Theideal single-enzyme experiment combines the developments from

both fields, allowing single-enzyme measurements to be per-formed with a drastically improved SNR under biologically rele-vant conditions. These strategies will push single-enzymeexperiments to the next level and ultimately answer the questionif enzyme kinetics is or is not characterized by dynamic disorder.

Acknowledgements

The authors thank Johan Hofkens, Chun-Biu Li and Tatyana Ter-entyeva for many fruitful discussions. This work was supported bythe Dutch National Research School Combination Catalysis Con-trolled by Chemical Design (NRSCC grant 2009-10016B, A.E.R.),The Netherlands Organisation for Scientific Research (NWO; VIDIgrant 700.58.430, K.B.; Gravitation program 024.001.035) and theHuman Frontier Science Program Organization (HFSP; Long-TermFellowship LT00866/2007-L, K.B.; Young Investigator GrantRGY0058/2010, K.B.).

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Single-enzyme kinetics with fluorogenic substrates: lessons learnt and future directions1 Introduction2 Single-turnover detection3 Single-molecule study of α-chymotrypsin4 Next-generation fluorogenic substrates5 Nanophotonic structures for single-enzyme experiments5.1 Zero mode waveguides5.2 Fluorescence enhancement by metal nanostructures5.3 Optical nanoantennas

6 ConclusionsAcknowledgementsReferences