ethane-bridged bis-porphyrin conformational changes as an effective

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ETHANE-BRIDGED BIS-PORPHYRIN CONFORMATIONAL CHANGES AS AN EFFECTIVE ANALYTICAL TOOL FOR NONENZYMATIC DETECTION OF UREA IN THE PHYSIOLOGICAL RANGE. Alessandro Buccolieri, Mohammed Hasan, Simona Bettini, Valentina Bonfrate, Luca Salvatore, Angelo Santino, Victor Borovkov, and Gabriele Giancane Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01230 • Publication Date (Web): 04 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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Analytical Chemistry

ETHANE-BRIDGED BIS-PORPHYRIN CONFORMATIONAL CHANGES AS AN EFFECTIVE ANALYTICAL TOOL FOR NONENZYMATIC DETECTION OF UREA IN THE PHYSIOLOGICAL RANGE. Alessandro Buccolieri,† Mohammed Hasan,‡ Simona Bettini,§ ± Valentina Bonfrate,§ Luca Salvatore, § Angelo Santino,# Victor Borovkov*,‡,⊥ Gabriele Giancane*||, ± †

Department of Biological and Environmental Sciences and Technologies (DiSTeBA), Università del Salento, Via per Arnesano, Lecce, Italy ‡

Department of Chemistry and Biotechnology, Tallinn University of Technology, Akadeemia tee 15, Tallinn, Estonia

§

Department of Engineering for Innovation, University of Salento, Via Per Arnesano, Lecce, Italy # Institute of Sciences of Food Production, C.N.R., Unit of Lecce, via Monteroni, 73100 Lecce, Italy ⊥

College of Chemistry and Materials Science, South-Central University for Nationalities, 182# Minzu RD, Hongshan District, Wuhan, Hubei province, 430074, China || Department of Cultural Heritage, Università del Salento, Via D. Birago, Lecce, Italy ±

UdR INSTM of Lecce University of Salento Via Monteroni, 73100 Lecce (Italy)

Corresponding authors’ email addresses: [email protected]; [email protected]

KEYWORDS Ethane-bridged bis-porphyrins, conformational molecular switching, host-guest interaction, urea detection, non-enzymatic sensing ABSTRACT: Conformational switching induced on ethane-bridged bis-porphyrins was used as a sensitive transduction method for revealing the presence of urea dissolved in water via non-enzymatic approach. Bis-porphyrins were deposited on solid quartz slides by means of the spin-coating method. Molecular conformations of Zn and Ni mono-metallated bis-porphyrins were influenced by water solvated urea molecules and their fluorescence emission was modulated by the urea concentration. Absorption, fluorescence and Raman spectroscopies allowed the identification of supramolecular processes which are responsible for host-guest interaction between the active layers and urea molecules. A high selectivity of the sensing mechanism was highlighted upon testing the spectroscopic responses of bis-porphyrin films to citrulline and glutamine used as interfering agents. Additionally, potential applicability was demonstrated by quantifying the urea concentration in real physiological samples proposing this new approach as a valuable alternative analytical procedure to the traditionally used enzymatic methods.

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Nitrogen containing compounds including ammonia and amino acids, which are not used in the protein synthesis, are metabolized and converted to urea molecules in the liver of living organisms. Urea is a nontoxic compound used by the body to process and to eliminate the nitrogen excess.1 Therefore, abnormal values of urea concentration in the blood is an indirect confirmation of several pathologies. In fact, an excess of urea in the blood, higher than 6.7 mM,2 can be due to a low blood flow towards the kidneys as a consequence of cardiac dysfunctions,3 chronic stress,4 bleeding from the gastrointestinal tract,5 high protein diet or dehydration.6 On the other hand, low urea concentration in blood suggests malnutrition,7 severe liver disease8 or it can be observed in normal pregnant patients.9 Conventionally, control and monitoring of urea level both in blood and in urine are performed in clinical conditions by means of enzymatic methods. The enzymatic approach uses the urease enzyme 10-12 to metabolize the urea molecules and to convert it into ammonia, that can be revealed with easy methods based, for example, on pH measurements 13 or color variation.14 Although the enzymatic method allows the detection of small amount of urea, many parameters have to be accurately controlled in order to preserve the enzymatic activity.15 In the present work, an alternative nonenzymatic approach of urea sensing on the basis of hostguest interaction followed by spectroscopic monitoring is proposed. Particularly, the intramolecular conformational switching of two metallated bis-porphyrin films induced by urea in a water solution was used as an effective analytical tool for its detection in the physiological concentration range. These conformational changes on the active molecules can be conventionally monitored by means of UV-Vis and fluorescence spectroscopies making the proposed method conveniently simple and rapid. Furthermore, its applicability for urea detection in real urine samples proposes the present new approach as an alternative procedure to enzymatic methods. EXPERIMENTAL SECTION Six different ethane-bridged bisporphyrins (1-6) 16,17 (in the SI file details of the synthetic procedure of 2 and 4 from 6 and 2, respectively, are reported) were used to study the possibility to reveal the presence of urea in aqueous solutions in the physiological range of concentration. These porphyrin hosts are of the same structural motif, whilst distinguishing by various combination of metal containing and free base porphyrin subunits. Previously, it was found that zinc and free base porphyrins are active species for the host-guest interactions in these bis-porphyrins as in solution 18,19 and in solid state.20,21 However, nickel containing porphyrins and bis-porphyrins are essentially inert in solution phase,17 whilst have not been tested in solid state. In the scheme 1, the chemical structures of the bis-porphyrins are sketched.

Scheme 1. a) Chemical structures of the ethane bridged bis-porphyrins (1-6) used in this study. For clarity, central atoms are labeled with the letters X and Y. b) aqueous analytes screened in the study. The chloroform solution of porphyrins (1-6) in the concentration of 7∙10-4 M were transferred on solid glass and silicon substrates by means of the spin-coating method. According to our previous studies,20 the spin rate and temperature influence the molecular arrangement of bis-porphyrins, changing it from syn- to tweezer and anticonformer (Scheme 2).

Scheme 2. Representative (a) Syn-, (b) tweezer and (c) anti-conformers of a generic bis-porphyrin. X and Y represent two metal ions or 2H in the case of free base porphyrin. A spin rate of 3200 rpm was used to transfer the bisporphyrins layer and the molecular arrangement after the deposition and the interaction with urea was monitored by means of a Shimadzu UV-2600 UV-VIS spectrophotometer. Fluorescence of the bis-porphyrins solid films was analyzed by a Shimadzu RF-5301 PC in the range 600 – 700 nm (excitation wavelength was set for each measurement according with the maximum absorption of the deposited film). FT-Raman spectroscopy (JASCO RFT-6000), equipped with a 1064 cm-1 laser, was used to monitor the variations of the molecular vibrational energies induced by the urea analyte on the bis-porphyrins solid films. The total amount of urea in the real samples was quantified by means of a colorimetric enzymatic test (purchased from Sigma Aldrich). It was obtained that 19 gr in 24 hours in a volume of 1.9 L of urea were produced, that corresponding to the urea concentration of about 0.166 M. All the reagents were purchased from Sigma Aldrich and used without further purification.


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Ultrapure water (MilliQ grade) was used to dissolve the analytes. RESULTS AND DISCUSSION One of the porphyrin main advantages as spectral sensors is its high extinction coefficient 21 allowing efficient monitoring of small physical-chemical changes by means of analyzing the corresponding electronic transitions in the UV-Visible range. 22-28 Additionally, the bisporphyrins with a flexible ethane linkage are able to switch the intramolecular conformation governing by host-guest interactions, hence generating distinguishable spectral outcomes.16 With this aim, spin coated films of bis-porphyrins (1-6) were deposited on quartz slides and the absorption spectra in the range of 350 – 650 nm were recorded. Subsequently, urea solutions with the concentrations between 0.01 mM and 15 mM were fluxed on the solid bis-porphyrin films and the eventual spectral changes induced by the analyte were monitored. These concentration limits were chosen in order to be compatible with the physiological range of urea in blood and urine.29 The contributions of both syn- and antiforms, respectively at 400 nm and 420 nm,30 are detectable when the porphyrin 1 is deposited as a solid film. The effect induced by fluxing 15 mM urea water solution on the absorption spectrum of 1 is negligible, as a result of insufficient host-guest interaction between the urea molecules and the porphyrin 1 film. Similarly, the porphyrin 6 exhibited invariant spectral profile upon the presence of urea, since the bis-porphyrin 6 exists exclusively in the corresponding anti – form. However, a more interesting spectroscopic behavior of both porphyrins 5 and 4 was observed. The spin coated film of porphyrin 5 is a mixture of the syn- and antiforms as in the case of Langmuir-Shäfer thin films31, while upon interaction with the urea solution (5 mM), this equilibrium is shifted to the anti- conformation (gray line in Figure 1a). This intramolecular switching induced by urea in the UV-Vis spectra appears even more evident when the host-guest interaction process is monitored by means of fluorescence spectroscopy with the excitation wavelength of 420 nm (Figure 1b). The significant enhancement of fluorescence emission is evident when the urea solution is fluxed on the 5 spin-coated film. A plausible rationale is that the fluorescence of syn- form is self-quenched by the close proximity of two Zn moieties owing to the strong π−π inter-porphyrin interaction,32 while the anti-conformer induced by the urea molecules ensures a larger distance between the two macrocycles and its unfavorable orientation for the non-radiative fluorescence decay, thus resulting in the emission enhancement of thin layer of 5. In the case of 4, the situation with the intramolecular spatial orientation is different yielding the anti-form exclusively upon deposition. It is worthy of note also that this bis-porphyrin has the same molecular geometry in nonpolar solvents that is in contrast to the synconformation of 5. A small bathochromic shift of the 4 absorption maximum upon interaction with urea is apparently a consequence of the ligation process of the guest molecule with the Zn moiety,20 whilst the conformation remains in the anti-form (gray line in Figure 1c). The interaction with urea enhances the fluorescence emission of 4 film when the injected urea concentration

increases as well as observed in the case of 5 (Figure 1d). However, in this case, whilst the conformation retains, the reason of fluorescence firing is an external ligation resulting in the increased electron density of central Zn ion, and hence decreasing the corresponding electron donating ability of pyrrole nitrogen to core Zn ion in 4.33 Besides, a red shift of the emission maximum indicates the axial ligation of urea.

Figure 1. Effect of urea injection on visible (figures a and c) and fluorescence spectra (b and d) of 5 and 4 (boxes a-b and c-d respectively). Fluorescence changes were monitored in the range of urea concentration 0 - 15 mM (in particular: 0 mM, 0.05 mM, 0.1 mM, 1 mM, 5 mM, 10 mM, 15 mM urea aqueous solutions were fluxed on the bis-porphyrins films).

A totally different spectroscopic effect was observed upon fluxing urea on the 3 active layer. As reported in Figure 2, the high energy B (Soret) band is hypsochromically shifted with a clear isosbestic point indicating the presence of two conformers. This is a result of the conformational change from anti- to tweezer form of 3 upon interaction with the urea solution (the urea concentration range is comprised between 0.01 mM and 5 mM). According to the previous rationale,20 interaction of the urea molecule with both the Zn and H2 atoms of 3 induces the corresponding conformational change yielding the tweezer form. Fluorescence emission of 3 (λ exc=418 nm) is quenched by the presence of urea; that is a consequence of the closer proximity of the two macrocycles in the tweezer configuration. This spatial arrangement results in a stronger exciton interaction between the porphyrin moieties that causes the nonradiative fluorescence quenching.34



Figure 2. a) Visible spectra of 3 fluxing urea at different concentrations. b) Fluorescence quenching induced by the urea on the emission of 3 spin-coated film.

Similarly to 4, 2 molecules deposited on quartz substrates are arranged in the anti-form with the maximum absorption centered at 414 nm. Also, a progressive red shift of up to 6 nanometers for the urea concentration of 5 mM is observed when the bis-porphyrin 2 molecules interact with the fluxed analytes (Figure 3a) and two isosbestic points are clearly present at 370 and 432 nm. This evidence suggests that urea chelates nickel ion 35,36 from the inner side of the macromolecule and hence induces a larger distance between the two porphyrin moieties. It is also confirmed by the fluorescence emission of the 2 layer (Figure 3b). In fact, the emission intensity of the solid film irradiated at 415 nm increases along with the urea concentration: the fluorescence quenching effect induced by the nickel atom is attenuated when urea is interposed between the two porphyrin moieties and the excitonic interactions between H2 and Ni subunits are considerably reduced. Whilst from the sensor point of view these spectral changes are promising, the structural details of this host-guest interaction mode is yet to be analyzed.

Figure 3. a) Visible spectra of 2 fluxing urea at different concentrations (box a). b) Fluorescence enhancement induced by the urea on the emission of 2 spin-coated film.

Furthermore, the Stern-Volmer plots 37 obtained both for 2 and 3 (Figure 4), as a ratio of the fluorescence intensities of the layer as deposited (I0) and after fluxing urea (I) versus the analyte concentration, suggest that the fluorescence emission variations induced by the analyte are ruled, in both cases, by a static quenching or enhancement phenomenon.37 It is revealed that two different linear regions can be identified with an abrupt slope change at 0.1 mM. It is reasonable to assume that the first linear region, at lower concentrations, is a consequence of the fluorescence static quenching of the outer layers of the deposited film. However, the second area is associated with the interaction of active molecules with the analyte that is ruled by the diffusion of urea molecules in the inner layers of the spin-coated film hence limiting the fluorescence emission variations. Linear dependence of the fluorescence quenching and enhancement on the analyte concentration is particular promising for sensing application of these two active layers. In fact, the observed linearity allows a facile determination of the urea concentration by means of the interpolation of the linear regions.


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detection. No significant variations in the corresponding absorption spectra were observed when the samples were treated for five minutes at different temperatures, starting from 5 °C up to 60 °C (Figure S1). The intensity of emission spectra is slightly influenced only for temperatures higher than 50 °C (Table S1), suggesting the use of the sensing device in a wide interval of temperature conditions.

Figure 4. Stern-Volmer plots of a) 3 and b) 2. The linear dependence of fluorescence emission of spin-coated film and fluorescence values recorded for different urea concentration is evident for both the active layers analyzed. Low and high concentration regions for the two active layers were linearly fitted with the R-squares of a) 0.974 and 0.992 and b) 0.991 and 0.960 respectively.

Raman spectroscopy was used to further characterize the host-guest interaction between 2, 3 and urea in water solution of 15 mM (Figure 5). Both the bis-porphyrins show the typical vibrational spectra of tetrapyrrolic molecules and the urea signal centered at 1005 cm-1 detectable after the interaction with the active molecules.38 Regions comprised in the range 1540 – 1586 cm-1 and 1320 – 1370 cm-1 exhibit the typical vibrations of C-C, CN-C and C-C-N of porphyrin core.39 In particular, it is worth to note that the signals at 1326 cm-1 and 1330 cm-1 observed in the 2 and 3 Raman plots, respectively, are imputable to the C-N vibrations of porphyrin core and influenced by the porphyrin central metal.40,41 After the urea injection, the intensities of both signals are changed as a consequence of the interaction of the analyte molecules with the inner part of the porphyrins’ macrocycle. Also, the effect of urea on the porphyrin 3 vibrations at low wavenumbers is evident; in fact, after the analyte injection, the signals at 260 cm-1 and at 640 cm-1 are almost disappeared. These vibrational bands can be assigned to the out of plane mode deformations of the four pyrrolic rings of each porphyrin.39 The urea, as observed by UV-Vis and fluorescence spectroscopy, promotes the formation of tweezer configuration, in which the two macrocycles are in closer proximity that inhibits the socalled saddle-type distortion of porphyrin rings.39,42,43 The effect of temperature on the spectroscopic features of 2 and 3 films was investigated in order to evaluate the temperature range where the device can be used for urea

Figure 5. Raman spectra of a) 3 and b) 2 before and after fluxing urea water solution (15 mM).

In order to verify the effect of possible interfering molecules on the response of 2 and 3 spectral changes, the solutions of urea, citrulline, and glutamine were fluxed on both the active layer. The choice was dictated by the fact that citrulline and glutamine amino acids are present in urine of patients affected by gastrointestinal diseases and hepatic disorders, respectively.44,45 Therefore, a potential urea sensor should be inert to these concomitants. In fact, the presence of the two amino acids does not influence the fluorescence response of the two active layers, and hence the urea concentration can be obtained by means of the calibration graphs according to Figure 4 a and b for 3 and 2 respectively (Table 1). This high selectivity further suggests prospective application of the proposed active layers for urea sensing by non-enzymatic approach. Table 1. Calculated urea (Urcalc) concentration in aqueous solutions containing citrulline (Citr) and glutamine (Glut) as interfering agents. The error reported for the calculated urea concentrations were obtained as a standard deviation on five measurements carried out on the same solution. [Ur] / mM


[Citr] / mM

[Glut] / mM

[Urcalc] / mM with

[Urcalc] / mM with 2


Sol 1 Sol 2 Sol 3 Sol 4

3 0.06 ± 0.03

0.07 ± 0.05

0.07

1

1

3

10

10

3.2 ± 0.2

2.9 ± 0.4

3

0

5

2.8 ± 0.3

2.9 ± 0.3

12

5

0

11.8 ± 0.3

11.3 ± 0.4

Furthermore, the applicability of these new sensors was nicely demonstrated by urea quantification in real physiological urine samples, which was carried out by the active layers 2 and 3 and compared with the results obtained by a clinical laboratory using traditional enzymatic method. The urea concentration obtained by means if enzymatic method was 0.166 M. For the sensing experiments with 2 and 3 active layers, the urine sample of a healthy male patient in the age group of 35-45 was used. The sample was centrifuged for 10 minutes at 2000 rpm and the supernatant was recovered. Then, the sample was diluted 20 times in ultrapure water in order to obtain a concentration of 8.3 mM and then it was fluxed on 2 and 3 spin coated films. This dilution procedure is necessary to ensure the linearity of spectral response of the sensing devices as highlighted in Figure 4. In fact, for the urea concentration higher than 25 mM the linear relationship between the I0/I value and the analyte concentration is affected. Apparently, at the high concentrations, all the active sites of 2 and 3 layers are involved in the interaction with the urea molecules and further analyte injection does not induce any appreciable spectral variation. Fluorescence variations were evaluated and the urea concentration was estimated on the basis of sensors’ responses (as reported in the figures 4a and 4b). In figures 6a and 6b the corresponding emission changes are plotted. The urea concentrations obtained from enzymatic method and from interpolation of the calibration curves are in a good agreement. In fact, the active layers 2 and 3 showed the responses corresponding to the urea concentration (7.92±0.38) and (8.61±0.34) mM, respectively, whilst the concentration determined by the enzymatic method was 8.3 mM.

Figure 6. Fluorescence changes of a) 2 and b) 3 induced by fluxing urine sample.

Meanwhile, the presence of ammonia in real urine samples could influence the conformational arrangement of bis-porphyrins via competitive binding, hence affecting the overall sensing response. In order to verify this rationale, solutions of urea and ammonia at different dilutions were fluxed on the active layers (Table 2). Same urea/ammonia ratio (corresponding to about 5:1) that is usually found in urine of healthy patients was adopted to test the interfering role of ammonia in the sensing process (ammonia is usually present in urine in a concentration of 0.03 M). As one can see, the urea presence was systematically overestimated by active layer 3 and underestimated in the case of bisporphyrin 2. As already reported,20 NH3 induces the conformational switching of the bisporphyrin 3 molecules towards the syn-conformer resulting in quenching of the fluorescence emission. In contrast, the emission enhancement in the 2 active layer is a consequence of the larger distance imposed by the analyte’s molecule between the two porphyrin moieties. Apparently, NH3 competes with urea in the interaction with the 2 molecules reducing the enhancing effect on the emission intensity. Although both the active layers detected the urea concentration in good agreement with the standard enzymatic method, it is worth to note that an opposite systematic deviation of almost the same percentage from the effective urea concentration is measured. Interestingly, whilst the response of active layer 2 at the 8 mM urea concentration appears to be reliable, its accuracy noticeably decreases upon dilution. However, at the same time, a slight over estimation is observed when 3 is used for the urea analysis in real samples. Thus, considering the statistics of our data with a clear systematic difference in the results as a possible consequence of contribution of interfering ammonia, 46 the average results obtained from the use of both the porphyrins (2 and 3) could give a fairly high accuracy of about 97-100%, even at the low concentrations (Table 2). Although the (supra)molecular and photophysical mechanisms of these sensors are yet to be comprehensively investigated, apparently the difference of host-guest binding in 2 and 3 results in


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certain molecular arrangements of the bis-porphyrin structures leading to the opposite tendency in the corresponding emissions. In turn, this is responsible for the systematic deviations observed in these sensors. So, the simultaneous use of 2 and 3 active layers in a sensor array could be a powerful tool to detect urea in real sample with high accuracy. Furthermore, the easiness of operation and quick response time potentially makes the proposed approach a valuable alternative to existing enzymatic methods.



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Table 2. . Effect of on calculated urea (Urcalc) concentrations (mM) in aqueous solutions containing additional NH3 solutes. The %[Urcalc] represent percentage (over and under) estimation of urea analyte using corresponding individual porphyrins, whereas average %[Urcalc] and [Urcalc] / mM represent average results obtained from both sensor 2 and 3 porphyrins. The error reported for the calculated urea concentrations were obtained as a standard deviation on five measurements carried out on the same solution.

[Ur] / mM

[NH3] / mM



%

%

2

Sol 5

8

1.5

7.87 ± 0.35

8.27 ± 0.43

Sol 6

1.5

0.3

1.31 ± 0.28

Sol 7

0.032

0.006

0.022 ± 0.03

3

Avg % 2+3

Avg / mM 2+3

98

103

100

8.07

1.67 ± 0.41

87

111

98

1.49

0.038 ± 0.02

67

126

97

0.03


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CONCLUSIONS The conformational switching induced by urea aqueous solutions on spin-coated films of six different ethanebridged bis-porphyirins was monitored by spectroscopic techniques. It was highlighted that the porphyrin metal central plays a crucial role in the interaction with the analyte molecules and it allowed us to identify the monometallated Ni and Zn bis-porphyrins (2 and 3 respectively) as active molecules for detecting urea in the physiological concentration range. The interaction between 3 and water dissolved urea induces the conformational change from anti- to tweezer form and the fluorescence emission is quenched by the presence of urea. The effect of the analyte on UV-vis spectrum of 2 is a red shift of the maximum absorption wavelength, whilst the fluorescence quenching effect induced by the nickel atom is attenuated when urea is interposed between the two porphyrin moieties. The linearity of the Stern-Volmer plot for both the active layers allowed to propose them as an effective analytical tool for detecting urea in water solution. The interfering role of citrulline and glutamine as possible concomitant compounds have been evaluated and a high selectivity of the sensing mechanism was highlighted. Finally, the active layers response was evaluated for urine real physiological samples. The urea quantification obtained using bis-porphyrins 2 and 3 was compared with that attained by a certified clinic analysis laboratory that used an enzymatic protocol. A good agreement between the produced results is extremely encouraging for the optimization of a prototype sensor device for application on urine samples.

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