Dual‐Color Real‐Time Chemosensing of a Compartmentalized Reaction Network Involving Enzyme‐Induced Membrane Permeation of Peptides

The design of synthetic systems with interrelated reaction sequences that model incipient biological complexity is limited by physicochemical tools that allow the direct monitoring of the individual processes in real‐time. To mimic a simple digestion‐resorption sequence, the authors have designed compartmentalized liposomal systems that incorporate extra‐ and intravesicular chemosensing ensembles. The extravesicular reporter pair consists of cucurbit[7]uril and methylene blue to monitor the enzymatic cleavage of short enkephalin‐related peptides by thermolysin through a switch‐off fluorescence response (“digestion”). Because the substrate is membrane‐impermeable, but the dipeptide product is permeable, uptake of the latter into the pre‐formed liposomes occurs as a follow‐up process. The intravesicular chemosensing ensemble consists of i) cucurbit[8]uril, 2‐anilinonaphthalene‐6‐sulfonic acid, and methyl viologen or ii) cucurbit[7]uril and berberine to monitor the uptake (“resorption”) of the enzymatic products through the liposomal membranes by i) a switch‐on or ii) a switch‐off fluorescence response. The dyes are designed to allow selective optical excitation and read‐out of the extra‐ and intravesicular dyes, which allow for dual‐color chemosensing and, therefore, kinetic discrimination of the two sequential reactions.


Introduction
One challenge of systems chemistry addresses the design of complex reaction networks that mimic the origin of life. [1]An important aspect of biological complexity involves the compartmentalization of different compounds and their Scheme 1.A model for a simple digestion-resorption sequence consisting of the enzymatic conversion of a membrane-impermeable peptide to a smaller, permeable one (the "nutrient") and its subsequent resorption into the lipidic compartment.The scientific challenge is to monitor the coupled reactions in real-time by rational selection of compartmentalized chemosensing ensembles.
Typical bioactive analytes that can be targeted with tandem assays are amino acids, biogenic amines, peptides, nucleotide phosphates, neurotransmitters, and aromatic metabolites as well as drugs.They have, during the past 15 years, been used by others and us for analyte sensing, [7e,f,8d,12] enzyme recognition, [7e,f,8d,12a,c] chirality sensing, [13] enantiomeric excess determination, [14] enzymatic activity testing, [7e,8a,b,12a,15] activator [15h,16] and inhibitor screening, [17] high-throughput screening, [8b,18] membrane permeation, [8c,19] porin and porin mutant characterization, [8c,12c] porin blocker detection, [8c] chemosensor sensitivity and selectivity enhancement, [20] and membrane carrier screening. [21]erein, we applied this supramolecular sensing strategy to an out-of-equilibrium reaction network which entailed i) a compartmentalized vesicular system, ii) an enzymatic reaction taking place on the outside, and iii) the selective permeation of the enzymatic reaction product through the lipid bilayer membrane.Such coupled processes are omnipresent in relation to cellular functions such as oxidative phosphorylation, apoptosis, photosynthesis, and signaling.To the degree that the enzymatic product can be viewed as a "nutrient", this reaction sequence can be referred to as a mimic for a digestion-resorption sequence (Scheme 1).To achieve direct time-resolved monitoring of both processes, the enzymatic reaction and membrane permeation, we needed to develop a system composed of two chemosensing ensembles that would allow separate monitoring of the individual processes, one positioned in the extravesicular phase (reporting on the digestion) and another one in the intravesicular phase (reporting on the resorption).Finally, to suppress crosstalk in the fluorescence output signals, the analyte-responsive dyes in the chemosensing ensembles needed to be optimized to allow selective excitation and fluorescence read-out at significantly differing wavelength (dual-wavelength monitoring or dual-color chemosensing). [22]

Results and Discussion
Our idea to biochemically realize the digestion-resorption sequence shown in Scheme 1 was such that we aimed to iden-

S3
Thr-Gly-Ala-Phe-Leu-OH P3 Phe-Leu-OH tify membrane-impermeable peptide sequences that could be hydrolyzed by a particular protease and that would afford membrane-permeable products.19a] In the course of our search, we found that the two peptides S1 and S2 and their products P1 and P2 (Table 1) fulfill these characteristics, because the former two are impermeable and the latter two are permeable.We also established by supramolecular tandem enzyme assays that they could be interconverted by thermolysin, an endoprotease that cleaves peptides at the N-terminus of phenylalanine. [23]In essence, the enzymatic products P1 and P2 are short, positively charged (amidated) dipeptides closely related to others that have previously been found to be membrane-permeable. [24]The substrates S1 and S2 are membrane impermeable, due to their increased length.
When the C-terminus is not amidated, such as for substrate S3 and its product P3, the negatively charged carboxylate group prevents membrane passage for both peptides, but their susceptibility toward enzymatic digestion by thermolysin is retained; they were used as controls in our experiments.It should be noted that peptides P1-P3 are variants of enkephalins, a class of endogenous pentapeptides that target the opioid receptor.

Tandem Enzyme Assays
15a] However, the originally employed reporter pair for monitoring the enzymatic reaction, namely cucurbit[7]uril•acridine orange (CB7•AO), turned out to be incompatible with the liposomal systems, because acridine orange interacts with the lipids, resulting in unstable liposomal formulations.Because we needed to identify an alternative reporter pair, we grasped the opportunity to search at the same time for a long-wavelength absorption and emission dye (> 600 nm), which would allow read-out in a different spectral range than those of conventional dyes (400-600 nm).We found that methylene blue (MB) suits this purpose because it binds strongly to CB7 (K a = [1.1 ± 0.2] × 10 7 M −1 , see Figure S2, Supporting Information), shows a sufficient fluorescence enhancement factor upon CB7 binding (≈1.6), and absorbs ( max = 665 nm) as well as emits ( max = 685 nm) in the red.In fact, MB has been previously studied in relation to its binding to CB7, [25] with reports of comparable affinities.However, the longwavelength optical peculiarities of the CB7•MB complex have never been exploited, such as for the dual-wavelength monitoring introduced below.The affinity of both, substrates and products, to CB7 was first determined by competitive fluorescence titrations (Figure S3, Supporting Information) with the CB7•MB reporter pair, which afforded binding constants on the order of 10 3 -10 4 M −1 for S1-S3 and 10 6 -10 7 M −1 for P1-P3 (Table 2); UV-vis titrations afforded consistent values (Figure S5 and Table S2, Supporting Information).15a,26] According to the tandem assay principle, [7g,10a,12c,27] when thermolysin is added to a solution of S1-S3, a weaker competitor is converted into a three orders of magnitude stronger competitor (P1-P3), which results in a fluorescence decrease that in turn reports on the enzyme kinetics.The resulting product-coupled tandem assay with switch-off fluorescence response is illustrated in Figure 1.
The dependence of the initial reaction rates on varying substrate concentration displayed Michaelis-Menten behavior, with the expected saturation at high substrate concentration (Figure 1c and Figure S7b,d, Supporting Information).15a]

Tandem Membrane Assays
8d] The former a) Determined by supramolecular tandem assay at varying peptide concentrations, see Figure 1b and Figure S7a,c, Supporting Information; enzyme kinetic parameters were determined by nonlinear regression analysis (see Supporting Information); b) 5% error; c) Data in square brackets refer to values obtained with the CB7•AO reporter pair, see ref. [15a].
affords a fluorescence increase upon analyte binding (because MV acts as a strong fluorescence quencher) while the latter affords a fluorescence quenching due to displacement of the dye.
The two assays correspond to those used in the previously established FARMA method for measuring analyte permeability, [19a] which is illustrated in Figure 2. Accordingly, when the respective chemosensing ensembles are encapsulated inside liposomes, permeation of the peptides should result in a switch-on fluorescence response (for CB8•2,6-ANS•MV) or a switch-off one (for CB7•BE).Experimentally, the expected fluorescence responses were only observed for products P1 and P2, but neither for substrates S1-S3 nor for product P3 (Figure 2 and Figures S9-S13, Supporting Information).This signaled that only P1 and P2 are membrane-permeable, as we had envisaged.
The time-dependent fluorescence changes progressed until a plateau was reached (Figure 2b,e, and panel (a) in Figures S10-S13, Supporting Information).Towards the end, for the CB8/MV/2,6-ANS assay, Triton-X-100 (TX-100) was added to lyse the vesicles, which resulted in a strong fluorescence enhancement due to dilution.This surfactant addition is routinely used to normalize the fluorescence recovery to its maximum.For the CB7/BE assay, adamantylamine (as a strong competitive binder) was added together with Triton X-100 to additionally ensure quantitative displacement of BE from CB7.Initial permeation rates were assessed [28] and plotted against the concentrations of the respective dipeptides P1 and P2 (Figure 2c,f, and Figures S9d,  S10d, S12d, and S13d, Supporting Information), which afforded the permeation rate constants (k p ) by linear regression analysis and, by considering the liposome radius, the related permeability coefficients (P), see Table 4.Note that the quantification of membrane permeability of peptides presents a standing challenge; existing methods are mainly based on planar lipid membranes and droplet interface bilayers or they are indirect and do not provide real-time data. [29]In addition to the various methods and lipid assemblies, existing data involve largely varying lipid compositions, which limits the direct comparison of absolute literature values, including the data in Table 4.

Optical Characteristics for Dual-Color Sensing
The unique feature of our compartmentalized chemosensing systems is that the two fluorescent dyes can be selectively excited and read out in a dual-color mode.The photophysical characteristics of the systems constituted of the extravesicular CB7•MB pair and the intravesicular CB8•2,6-ANS•MV ensemble are shown in Figure 3a.As can be seen, MB should allow for a specific excitation at 665 nm and an unperturbed observation by fluorescence at 685 nm ("MB channel").In contrast, 2,6-ANS can be excited at 317 nm and it emits in the spectral window at 476 nm ("2,6-ANS   4. Membrane permeation rate constants, k p , and permeability coefficients (P), of peptides, determined by the FARMA method [19a] in POPC/POPS liposomes.channel").The spectral properties of the second system, which incorporates the extravesicular CB7•MB pair with the intravesicular CB7•BE one, are shown in Figure 3b.As can be seen, BE can be selectivity excited at 420 nm and followed by its 495 nm fluorescence ("BE channel"), well below and not overlapping with the long-wavelength MB absorption and emission.Since the extravesicular chemosensing ensemble reports on the enzymatic conversion while the intravesicular one signals the transmembrane permeation process, and since both sensors can be selectively sounded out in dual-color mode, it should become possible to monitor the two sequential "digestion-resorption" processes directly in the same sample.

Dual-Color Chemosensing in Coupled Tandem Enzyme-Membrane Assays
With the substrate/product peptide pairs and their differential affinities (Tables 1 and 2, and Tables S1 and S2, Supporting Information), the individual tandem assays (Figures 1 and 2 as well as Tables 3 and 4), and the spectroscopic characteristics of the dualcolor chemosensing ensembles (Figure 3), we had the required tools in hand to proceed to the next level of complexity and merge tandem membrane and tandem enzyme assays into a single system.The working principle of the coupled tandem enzymemembrane assays is shown in Figure 4.For this purpose, liposomes with the respective encapsulated permeation-responsive chemosensing ensembles, CB8•2,6-ANS•MV and CB7•BE, were prepared and the enzymatic reaction-responsive reporter pair, CB7•MB, was added to the outside solution.The two systems (Figure 4a,c) only differ in the selection of the intravesicular reporter pair, the enzymatic reaction is monitored in both cases with the CB7•MB reporter pair, which affords a switch-off fluorescence response when monitored in the MB channel.For the system with the intravesicular CB8•2,6-ANS•MV chemosensing ensemble (Figure 4a), a switch-on fluorescence response is expected to result from the subsequent analyte permeation (in the 2,6-ANS channel), while for that utilizing CB7•BE, a switch-off response (Figure 4c) is expected to become observable in the BE channel.
Numerous control experiments were performed first.In particular, we demonstrated that the addition of substrates S1-S3 to the dual-chemosensor, dual-compartment system in the absence of enzyme did not trigger any time-resolved MB fluorescence changes for the extravesicular reporter pair (except for a small "instantaneous" step indicative of weak binding) and that the fluorescence of 2,6-ANS or BE, which were in a different compartment inside the liposomes, remained unchanged (Figure S14, Supporting Information).This provided evidence that the substrate did not permeate through the liposomal membranes.Also, when MB (the only reporter pair component whose permeability had not been previously tested) was added to the bulk, no fluorescence change of the intravesicular reporter was observed (Figure S15, Supporting Information), confirming that this dye did not permeate through the lipid bilayer membranes on its own.In contrast, the addition of the products (P1-P3), in the presence of an enzyme, produced a time-resolved fluorescence response for products P1 and P2, indicative of fast membrane permeation (Figures S16-S19, Supporting Information), but no change for product P3, as it had been found to be impermeable.The permeation kinetics was virtually the same as for the absence of thermolysin (comparing Table 4 and Table S3, Supporting Information), which showed that the enzyme did not interfere with the permeation process of the dipeptide products.We also verified that the permeation kinetics monitored with the two intravesicular chemosensing ensembles in the absence of the extravesicular reporter pair (Figures S23 and S24, Supporting Information) was comparable to that observed in its presence (Figure 4b,d, and  Figures S20a and S21a, Supporting Information).8d] In fact, we also performed the permeation experiments by direct addition of the products P1 and P2 but with CB7/MB outside of the liposomes (Figures S25 and S26, Supporting Information)  and obtained comparable permeation rates as in the absence of the extravesicular reporter pair, which clearly shows that CB7 and MB outside of the liposomes do not influence the product permeation (Table S5, Supporting Information).
After the control experiments had been successfully performed, the coupled tandem enzyme-membrane assays were conducted with all components present in their respective compartments (extravesicular: thermolysin, CB7•MB; intravesicular: CB8•2,6 ANS•MV or CB7•BE) and the reaction network was initiated by addition of substrates S1-S3 (Figure 4 and Figures S20-S22, Supporting Information).Substrates S1 (Figure 4b,d) and S2 (Figures S20a and S21a, Supporting Information) afforded the expected fluorescence decline in the MB channel (red traces), indicative of the enzymatic cleavage of the pentapeptide to the dipeptide by thermolysin and the decay kinetics increased with substrate concentration, as expected.In the 2,6-ANS channel, which signals membrane transport of the dipeptide products, a slower increase in fluorescence was observed (black traces in Figure 4b and Figure S20a, Supporting Information), which also depended on the initially added substrate concentration.This experiment presents likely the best "visualization" of the digestion-resorption process because the digestion/depletion is echoed by a switch-off response, while the resorption/uptake is signaled by an opposite one, a switch-on.The fact that the membrane permeation kinetics was significantly slower than the rate of the enzymatic reaction showed that the former process was rate-determining under the selected reaction conditions.
When the same reaction sequence was initiated with the intravesicular CB7•BE reporter pair, both processes (enzymatic reaction and membrane transport) afforded a substrateconcentration-dependent fluorescence decrease in both, the MB and the BE channel (Figure 4d and Figure S21a, Supporting Information).Notably, the kinetics for the resorption step was also retarded, and the plateau region, which signals the completion of the process, was reached more readily for the MB channel responsible for sensing the substrate digestion than for the BE channel responsible for sensing the membrane permeation.For both set-ups, the addition of substrate S3, which produces the membrane-impermeable product P3, gave only a signal change in the MB channel for the enzymatic reaction, while the 2,6-ANS and BE channels remained silent (Figure S22, Supporting Information).
The two reactions in the compartmentalized tandem enzymemembrane assay system describe a simple reaction network of the sequential reaction type (A → B → C).Formally, product B is a "metabolite" that is first formed in the extravesicular space, while C is strictly speaking not a follow-up product, but a product in a different state/compartment.A quantitative description of the reaction network requires a set of coupled differential equations describing the relationship between the build-up of the enzymatic reaction product B and membrane permeation to the reach state C, as well as the different dye displacement processes.The situation simplifies (see Supporting Information), because, first, we can assume that the enzymatic reaction does not show sizable product inhibition and, second, that it is unaffected by the presence of the liposomes and their content.This was indeed confirmed when the enzyme kinetic traces (red traces in Figure 4b,d, and Figures S20a and S21a, Supporting Information) were analyzed with respect to their concentration dependence (Figure 4e,f, and Figures S20d and S21d, Supporting Information), which revealed that the resulting enzyme-kinetic parameters were very similar to those obtained in the absence of liposomes (comparing Table 3 with values in Tables 5 and Table S4, Supporting Information).Moreover, the kinetic analysis of stepwise enzymatic analyte formation and subsequent membrane permeation can be simplified by an initial rate approach.Therefore, the inverse initial permeation rates were multiplied with a fixed time interval, during which the permeation rates were still linear, and plotted against the inverse of the initial substrate concentration (see Supporting Information for the derivation of the relationship).Regression analysis of the obtained plots (Figure 4e,f, and Figures S20d and S21d, Supporting Information) afforded the permeation rates and permeation coefficients for the coupled tandem assay system (k P and p-values in Table 5), which nicely matched  c) Values in square brackets refer to the system with intravesicular CB8•MV•2,6-ANS sensing ensemble (instead of CB7•BE), note that this reporter pair affords systematically too low permeation parameters, which, however, are internally consistent, see Table 4.
the values determined by direct addition of the product peptides for the respective chemosensing ensemble (Table 4).The outcome of this kinetic analysis has two implications.First, it suggests that the permeation process, while coupled to the preceding enzymatic reaction, proceeds with the same (apparent) kinetics despite the multiple components in the system and their interrelated interactions, reactions, and transport processes.Moreover, it suggests that the extravesicular concentration of the permeating analyte (product B in the reaction sequence A → B → C) is significantly built up and that permeation of the analyte to reach state C proceeds with some delay.The compartmentalized reaction network may thus be considered as a first step towards a simple out-of-equilibrium reaction network of the sequential reaction type. [30]Importantly, the congruence between the membrane kinetic parameters in the direct permeation experiment (Table 4) and the sequential dual-compartment experiment (Table 5) indicates that the entire reaction network remains fully kinetically predictable and stable.
These mechanistic implications were further confirmed by a dual-compartment experiment, in which the enzyme concentration was varied, while the substrate concentration was kept constant (Figure S27, Supporting Information).With decreasing enzyme concentration, the initial permeation rates approached the initial rates for enzymatic conversion, which is fully consistent with a gradual change of the rate-determining step; at high enzyme concentration, membrane permeation is rate-limiting, while the biocatalytic reaction becomes rate-limiting at low enzyme concentration.Although we focused here on the former scenario (Figure 4), since it allowed the dissection of the two sequential reaction steps (Table 5), these detailed observations are conceptually intriguing and clearly demonstrate the strength of the dual-color chemosensing approach to resolving simultaneous reaction steps in complex supra-biomolecular reaction networks.

Conceptualization
As we advance the utilization of tandem assays to higher levels of complexity, it is timely to categorize the principal variants (Figure 5).The original variant, tandem enzyme assay, was designed for the monitoring of a biocatalytic reaction (A → B) in the presence of a single reporter pair (chemosensor I) in a homogeneous solution.The second principal version, tandem membrane assay, has been developed for following membrane permeation (B → C) by encapsulating a single reporter pair (chemosensor II) in the inside of vesicles or cells.The sensor system we introduced here couples the two assay methods (tandem enzyme-membrane assay) and can follow a sequential process A → B → C in the presence of two spatially separated reporter pairs (chemosensors I and II).To assemble the two reporter pairs, 4-5 concentrations, of 2 macrocyclic receptors and of 2-3 guests (dyes), need to be carefully adjusted.These components, as well as their complexes, must be membrane-impermeable.Moreover, the two dyes need to be optically complementary to allow both processes to be differentiated.

Conclusion
We have investigated a system with spatially and optically separated, compartmentalized chemosensors that has allowed us to mimic and temporally resolve two fast reactions in a simple peptide digestion-resorption network.By using a tandem enzymemembrane system, the biochemical reaction sequence became optically observable and could be kinetically resolved in both compartments (Figure 4b,d and Figures S20a and S21a, Supporting Information).The method can eventually be extended to mimic related functional cellular pathways, such as signaling, or, more generally, to logic-gate operations. [31]The compartmentalization of chemosensors itself does not only allow for a methodological variation but it can be exploited practically for sensing.On one hand, through the compartmentalization of one reporter pair, its interference with the outside enzymatic reaction, for example, an inhibition, as well as crosstalk between two reporter pairs is effectively prevented.26b] was diluted to 2 mL with (NH4)2HPO4 buffer (10 mM), pH 7.0 to afford a final phospholipid concentration of 25 μM.The cuvette was equilibrated at (25.0 ± 0.1) °C and the fluorescence intensity was recorded ( exc/em = 317/476 nm for CB8/MV/2,6-ANS and  exc/em = 420/495 nm for CB7/BE) while gently stirring.Subsequently, the dipeptide was added at t = 60 s.For the CB8/MV/2,6-ANS assay, 24 μl 1.2% (wt/vol) Triton X-100 in water was added at the end of the experiment to lyse the vesicles, for calibration.For the CB7/BE assay, 40 μM adamantylamine (as a strong competitive binder) was added together with 24 μl 1.2% (wt/vol) Triton X-100 in water at the end of the experiment to i) lyse the vesicles and ii) to ensure concomitant quantitative berberine displacement from CB7.In the CB8/MV/2,6-ANS assay, time courses of I t were normalized to I norm by using the equation I norm = (I t − I 0 ) / (I ∞ − I 0 ), where I 0 = I t before peptide addition and I ∞ = It after lysis.This equation afforded time traces, where the intensity was zero before addition and 1.0 when 2,6-ANS was completely displaced from CB8/MV.In the CB7/BE assay, normalization of I t was performed by using the equation I norm = (I t − I ∞ ) / (I 0 − I ∞ ), where I 0 = I t before peptide addition and I ∞ = I t after lysis.This equation afforded time traces, where the intensity was 1.0 before addition and zero when BE was completely displaced from CB7.
Dual-Color Tandem Enzyme-Membrane Assay: In a typical dual-color experiment, liposome stock solution (20 μL) loaded with CB8/MV/2,6-ANS or CB7/BE was diluted to 2 mL with (NH4)2HPO4 buffer (10 mM), pH 7.0, containing CB7 (1 μM) and MB (0.5 μM) to afford a final phospholipid concentration of 25 μM.The cuvette was equilibrated at 25.0 ± 0.1 °C and the fluorescence intensity was recorded in a dual-channel mode as a function of time while gently stirring ( exc/em = 665/685 nm for CB7/MB,  exc/em = 317/476 nm for CB8/MV/2,6-ANS, and  exc/em = 420/495 nm for CB7/BE).Subsequently, first thermolysin (0.063 U mL −1 ) was added at t = 60 s to the solution and then the substrate at t = 120 s.At the end of the experiments, Triton X-100 with or without adamantylamine was added, for the tandem assay (see above).Two different kinetic traces were obtained simultaneously in the dual-channel mode to monitor the enzymatic conversion and membrane permeation processes separately.The time courses of I t were normalized as described in the preceding sections.
Statistical Analysis: Assay results were obtained in triplicates and data normalization was performed as described for the individual assays in Methods.Reported errors refer to SD (±).

Figure 1 .
Figure 1.a) Working principle of a fluorescence switch-off tandem enzyme assay using CB7 and MB as reporter pair.b) Continuous fluorescence enzyme assay for S1 with the CB7•MB reporter pair (0.063 U/mL thermolysin, 0-20 μM S1, 0.5 μM MB, and 1 μM CB7 in 10 mM (NH 4 ) 2 HPO 4 , pH 7.0, at 25 °C;  exc = 665 nm,  em = 685 nm.c) Dependence of the initial rates of the enzymatic reaction on S1 concentration.Statistical errors in (c) are within the size of the data points.

Figure 3 .
Figure 3. Dual-color detection with two different reporter pair combinations: a) CB8•2,6-ANS•MV and CB7•MB, and b) CB7•BE and CB7•MB.The normalized absorption (solid) and emission (dashed) spectra show selective reporter pair monitoring without spectral crosstalk between the channels.BE and MB spectra were recorded at 0.5 μM in the presence of 1 μM CB7, and 2,6-ANS spectra at 2 μM in the presence of 2.5 μM CB8 and MV.

Figure 4 .
Figure 4. Operational principle for dual-color chemosensing of a reaction network involving an enzymatic reaction coupled with permeation of the formed product.In both implementations, thermolysin (0.063 U mL −1 ) activity is monitored with the CB7•MB (CB7: 1 μM, MB: 0.5 μM) reporter pair outside the liposomes in 10 mM (NH 4 ) 2 HPO 4 at pH 7.0, at 25 °C, and, simultaneously, membrane permeation of the enzymatic product (dipeptide) is monitored in the inside of the liposomes with different chemosensing ensembles.a) The CB8•2,6-ANS•MV chemosensing ensemble (CB8: 500 μM, 2,6-ANS: 550 μM, MV: 550 μM) is encapsulated in liposomes in 10 mM Hepes buffer, pH 7.0, at 25 °C, and permeation of the dipeptide product leads to binding to the receptor which results in a switch-on fluorescence response.b) Changes in the (red) MB emission intensity ( exc = 665 nm,  em = 685 nm) and (black) 2,6-ANS emission intensity ( exc = 317 nm,  em = 476 nm).c) The reporter pair CB7•BE (CB7: 500 μM, BE: 550 μM) is encapsulated in liposomes in 10 mM (NH 4 ) 2 HPO 4 buffer, pH 7.0, at 25 °C, and permeation of the dipeptide product leads to binding to the receptor CB7, which results in a switch-off fluorescence response.d) Changes in the (red) MB emission intensity ( exc = 665 nm,  em = 685 nm) and (black) BE emission intensity ( exc = 420 nm,  em = 495 nm).Double-reciprocal dependence of the initial permeation rates on substrate concentration (top) and dependence of initial enzymatic reaction rates on substrate concentration (bottom) for the system with e) intravesicular CB8•2,6-ANS•MV and f) CB7•BE chemosensing ensemble.

Figure 5 .
Figure 5. Assay categorization: a) Tandem enzyme assay.b) Tandem membrane assay.c) Tandem enzyme-membrane assay.A-C are analytes (states) and I and II are chemosensors, typically composed of macrocycle and dye.

Table 2 .
Affinity constants, K a , of CB7 with the investigated peptides.

Table 3 .
Enzyme-kinetic parameters for the conversion of peptides S1-S3 by thermolysin.