GB2279739A - Porphyrin sensors - Google Patents

Abstract

Sensors comprises a monomolecular layer of a tetra(alkylsulphonamido) phenyl porphyrin. Absorption into the layer of a substance to be detected causes a variation in optical absorption which can be measured spectrophotometrically e.g. at 350 - 750 nm.

Description

PORPHYRIN SENSORS This invention relates to sensors comprising Langmuir monolayers of porphyrins.

Langmuir monolayers of porphyrin were first described by Alexander (J. Chem. Soc. 1937, p. 1813) and several papers have been published on the film forming and structural properties of porphyrins (e.g. Shick et al, J.

Chem. Soc. 111 1989 pp 1344-1350, Miller et al, Thin Solid Films 133 1985 pp 83 to 91, and Azumi et al, J. Chem. Soc.

114 1992 pp 10662-10663). Molecular interactions within such a monolayer can give rise to shifts or splitting in the absorption bands shown by such layers caused by exciton coupling (Kasha et al, Pure Appl. Chem. 11 1965 pp 371-392) or the formation of new molecular states (Ruadel-Teixier et al Thin Solid Films 99 1983 pp 371-392).

We have now found that certain substituted tetraphenylporphyrins produce Langmuir mbnolayers having novel absorption properties which make them suitable for use in sensors.

The tetraphenylporphyrin derivatives used in the present invention may be represented by the general formula:

wherein R is an aliphatic hydrocarbon radical containing a straight chain of at least 5 carbon atoms. Preferably R is a straight chain aliphatic radical of formula C,H2,1 where n is 7 to 20, and particularly preferred values of R are n-octyl (-C8 H17) and n-dodecyl (-C12 H25). The compound of formula I wherein R is n-dodecyl is herein after referred to as P1 and the compound of formula I wherein R is n-octyl is hereinafter referred to as P2.

Formula I as shown above is that of the free base (hereinafter abbreviated to PH2). This base is capable of forming a di-cation with 2 protons and this di-cation is hereinafter referred to as PH4++.

The tetraphenylporphyrins of formula I form Langmuir monolayers in which the optical absorption is very sensitive to changes in the separation of the molecules of the said porphyrin in the said layer. Such change may be brought out either by changing the degree of compression of the layer or by incorporation into the layer of other molecules. Furthermore, the optical absorption of such layers is very sensitive to pH as the separation of the molecules depends inter alia on the proportion of the molecules in the PH2 and PH4++ states.

The tetraphenylporphyrin derivatives of formula I may be prepared by reaction of an amine of formula RNH2 with tetra(4-chlorosulphonyl)phenylporphyrin, itself made by reaction of chlorosulphonic acid with tetraphenylporphyrin.

The present invention accordingly provides sensors comprising a monomolecular layer (a Langmuir monolayer) of a porphyrin of formula I, or a plurality of such layers, a support for the said layer or layers, which may be liquid, e.g. water, or solid, e.g. glass, and means for measuring variations in the optical adsorption of the said layer or layers in response to changes in the separation of the molecules of the said porphyrin in the said layer or layers brought about by adsorption into the said layer or layers of a substance to be detected.

The sensitivity of the layer to the substance to be detected depends on the pH at which the layer has been formed. As described in detail below, the pH critically affects the sensitivity of the layer and it is therefore important to optimise the pH at which the layer is formed.

This optimum PH is dependent inter alia upon the chain length of the radical R in the porphyrins of formula I. SYNTHESIS OF PORPHYRINS OF FORMULA I The starting materials used in the preparation of the substituted tetraphenylporphyrins of formula I were commercially pure compounds. All solvents were purified by the usual methods before use. Tetraphenylporphyrin (TPP) was prepared by the Rothemund method (Adler et al, J. Org.

Chem. 32 (1967), 476) and purified by the method of Barnett et al (J. Chem. Soc. Perkin I (1975) 1401). For the preparation of the required intermediate, meso-tetra(4 chlorosulphonyl)-phenylporphyrin, the TPP was reacted with ClSO3H and the product was isolated by precipitation with ice/water and filtration. The meso-tetra(4-aminosulphonyl)- phenyl-porphyrins of formula I were obtained by reaction of meso-tetra (4-chlorosulphonyl) phenylporphyrin with the corresponding amine of formula RNH2, in methanol, in almost quantitative yield in all cases. The porphyrins, which are not water-soluble were purified by precipitation in chloroform/hexane followed by flash chromatography on alumina and crystallization. They were characterized by FTIR and W-Vis spectroscopy, FAB/MS and microanalysis.

The free base porphyrins are reddish purple with an intense Soret band below 420 nm and four Q bands; when the central nitrogens are protonated the solution becomes bright green and a Soret band near 430 nm and a single Q band near 650 nm are observed. The spectra are similar in all cases with shifts of only a few nm being observed with different substituted groups R. This is explained by the lack of conjugation across the phenyl rings, which are probably almost orthogonal to the plane of the porphyrin ring itself. Solution spectra in various solvents for compound P1 are summarised in Table 1.

TABLE 1: Spectra of porphyrin P1 in various solvents.

Solvent | Soret Q bands (nm) band (nm) Alcohol 410 516 550 595 655 Acetone 418 512 545 590 660 Methanol 413 510 545 585 642 Methanol + TFA 437 - - - 650 Chloroform * 420 520 555 605 450 - - - 660 Dichloromethane 432 - - - 649 Floating layer * 421 515 550 590 640 487 - - - 608 Transferred layers ~ 380 440 These spectra correspond to the protonated and unprotonated states.

One of the characteristic features-of many free base porphyrins is strong fluorescence from the Q band. However, the present materials exhibit only very weak fluorescence, which occurs between 680 nm and 735 nm.

PREPARATION OF LANGMUIR FILMS 1.0-2.0 mg of the porphyrin of formula I is dissolved in 0.5 ml of methanol followed by the addition of 9.5 ml of chloroform (chloroform acts as a good spreading agent for distributing the porphyrin molecules over the water surface). Approximately 500 1 of this solution is then spread dropwise onto an ultra pure water surface contained within a Langmuir trough (Langmuir, J.Am. Chem. Soc., 39 (1917) 1848). Approximately 5 minutes is allowed for evaporation of the methanol and chloroform, and the floating film is then compressed using the Langmuir trough technique. This results in the formation of a floating porphyrin monolayer film.

Langmuir films were studied on a pure water subphase at 20-22 C and pH 5.8 unless otherwise stated. The films were transferred onto Corning 7059 glass and evaporated aluminium substrates at speeds of 1 mm/min for the first layer and 2-5 mm/min for subsequent layers.

An MCPD 100-311C spectrophotometer, manufactured by Otsuka Electronics, was used to record solution and film spectra. This offers a high speed sensitive system. Two interchangeable light sources were used; a deuterium lamp for Us studies and a tungsten filament lamp for visible spectra. The system is extremely flexible, since nonfluorescent low-loss quartz fibre bundles are employed to direct light to and from the sample. In the detector, a diffraction grating (133 lines per mm) is used to spectrally resolve the light onto a 1024-element photodiode array. This records the entire spectrum between 267nm and 1254nm with a resolution of 1.9 nm and sample times as short as 12 ms. Compensations were made before each reading for the dark-current of the photodiode array and the dc components of the analogue amplifier circuit.The system is controlled using a personal computer, which allows real-time analysis of the results.

The geometry shown in Figure 1 has been used in the characterisation of solutions, Langmuir-Blodgett films on optical glass (Corning 7059) slides, and thin-films filters. This technique can be extended to monitor the absorbance of a floating monolayer by positioning a mirror under the water surface. Changes in the absorption spectrum as the film is compressed can frequently be linked to phase changes in the r-A isotherm, providing further information about molecular orientation within the film.

The optical absorption band at about 490 nm is found for the C12 compound P1 only when pH < 3.8 and when the film is compressed. For the C8 compound P2, the situation is the same except that the threshold pH is 7.2. The intensity of this peak is dependent on the pH and on the area occupied by each porphyrin molecule (the degree of compression). Thus at any particular pH (provided pH < 3.8 for C12 porphyrin) the intensity of the peak gives a measure of the area per molecule (the degree of compression). If the porphyrin monolayer adsorbs a reasonably large molecule, e.g. from the water subphase, the porphyrin molecules separate in order to accommodate the adsorbate. Such separation leads to a reduction in the intensity of the adsorption peak.Thus the change in the intensity of this peak provides a means of detecting the uptake of the adsorbate, and therefore behaves as a sensor for presence of the adsorbate.

The porphyrins of formula I can exist in two states, namely as a free base (PH2) or as a dication (PH4++). The latter is produced by the addition of two protons into the central ring system. The optical absorption spectra of P1 and P2 in solution and in their free base state are characterised by a single band near 415 nm, termed the Soret band, together with four additional bands between 500 and 650 nm, termed the Q bands. The dication state is characterised by a Soret band near 445 nm and a single Q band near 645 nm. The exact positions of these bands depend on the particularly porphyrin and on the solvent.

The absorption spectra of P2 in dilute solutions in methanol are shown in Figure 2a for both the free base and dication states (formed by addition of TFA to the free base solution). For more concentrated solutions of P2 in methanol the absorption spectra of the free base state are similar to those in dilute solutions. However, the spectra of the dication show two extra peaks at 475 and 689 nm together with those of the normal state as seen in Figure 2b. Splitting of the Soret band due to exciton coupling has been reported previously (Osaka et al J. Amer. Chem. Soc.

110 (1988) pp 4454-4456). However, this is not usually accompanied by a similar splitting of the Q band (Schick et al J. Amer. Chem. Soc. 111 (1989) pp 1344-1350). The two extra peaks probably correspond to the formation of a new state which exists in equilibrium with the dication.

Similar effects can be seen with less concentrated solutions in methanol although this only occurs with the addition of more TFA.

This new state, herein called state X, appears to be produced by a combination of high concentrations and acidic conditions. The optical properties of both P1 and P2 on subphases with a range of values of pH have been investigated.

Figure 3a shows the r-A compression isotherms for P1 on subphases with different values of pH. For a subphase with pH 4.3 the isotherm shows a breakpoint at an area per molecule of 66 A2. Molecular models would predict an area of about 200 A2 for the porphyrin rings lying parallel to the water surface and about 50 A2 for the porphyrin rings lying perpendicular. Thus the rings may be in a tilted configuration with the breakpoint indicating a sudden change of this structure. As the pH of the subphase is reduced the isotherm takes on a much steeper nature and the breakpoint is less apparent. At a subphase pH of 2.85 the isotherm becomes very condensed which may indicate that the porphyrin molecules are existing in aggregated islands on the subphase surface. For this isotherm an extrapolated area per molecule is found to be 49.1A2.

When P1 is spread onto a subphase with pH 4.3 the absorption spectrum is that of the free base, regardless of its initial state when applied. The Soret band occurs at 420 nm. On compression the absorbance increases as would be expected owing to the increase of the surface density of chromophores, and there is a hypsochromic shift of the Soret band of the order of 9 nm. However, when P1 is spread onto a subphase with pH 3.45 two extra peaks at 490 and 700 nm develop on compression of the monolayer, together with that of the free base Soret band. These extra peaks can be attributed to the state X. The absorption spectra of P1 on a subphase with pH 3.45 for varying molecular areas are shown in Figure 4.It can be seen from Figure 4 that for molecular areas of less than 150 A2 the free base Soret peak does not increase with decreasing molecular area as would be expected for a purely density driven process.

Instead the intensity remains approximately constant and that of the state X increases. There is again a hypsochromic shift of the order of 9 nm for the Soret band peak. Between these two regions of subphase pH a switching point-exists in the production of the state X; where for floating layers on subphases with pH < 3-8 the state X can be formed, but for pH > 3.8 state X is not observed. Near this switching point the free base, dication and state X can all be seen simultaneously on the subphase surface as depicted in Figure 5. However, it is found that when the floating layer is kept confined to a constant surface area the absorption peak associated with the dication state quickly decays with time whereas that of state X increases.

The formation of the state X for floating layers is dependent on both the surface concentration of the porphyrin molecules and the acidity of the subphase. Figure 3b shows the ratio of the main peak of the state X at 490 nm to that of the Soret peak (PH2) for several values of the subphase pH. From this figure it can be seen that on very acidic subphases, pH 2.85, the peak at 490 nm dominates over the Soret band peak (i.e. X/PH2 is large) even when the molecules are well separated. However, as the pH of the subphase is increased towards the switching point, higher surface concentrations are needed to form state X. A general trend can also be seen whereby at high molecular areas the initial ratio of peaks increases for decreasing values of pH.

For floating layers of P1 on subphases with pH 2.85 the Soret peak occurs initially at 427 nm and undergoes a hypsochromic shift of only 2 nm on compression, indicating that exciton coupling is occurring. This differs from subphases with pH 3.45 where the peak is initially at 420 nm and a hypsochromic shift of 9 nm is measured on compression. This may indicate that a form of aggregation is occurring on the subphase surface for low values of pH and could explain the apparent fluctuations in the data for the ratio of peaks for pH 2.85. These fluctuations would then be due to inhomogeneities in the floating film which pass through the incident light beam causing larger/smaller absorptions to occur than would otherwise be expected.

Figure 6a shows the r-A compression isotherms for P2 on subphases with different values of pH. For a subphase with pH 6.78 a breakpoint in the isotherm can be seen similar to that for P1 although it occurs here at a lower surface pressure. As the pH is lowered the isotherms take on a less expanded appearance with the breakpoint becoming less apparent. The predicted extrapolated areas per molecule are seen to increase with decreasing values of the subphase pH. For a subphase with pH 5.70 the isotherm of P2 become very condensed and is almost identical to that of the longer chain derivative P1 on a subphase with pH 2.85.

The spectral properties of the floating layers of P2 were found to be very similar to that of P1 although the state X could be formed at much higher values of the subphase pH. For floating layers of P2 on subphases with a pH > 7.2 the porphyrin molecules remained in their free base state during compression of the monolayer. However, on subphases with pH < 7.2 the two extra peaks corresponding to the state X, occurring here at 480 nm and 699 nm, were again seen. The ratios of the state X peak at 490 nm to that of the free base peak, PH2, are shown in Figure 6b.

Monolayers of P1 and P2 can be transferred onto cleaned glass substrates by the vertical LB deposition method. If the monolayers of P1 are transferred from subphases with a pH > 3.8, the optical absorption spectrum of the LB film represents that of the free base porphyrin.

However, if the monolayer is transferred from a subphase with a pH < 3.8 then the optical absorption spectrum of the LB film shows both the free base state and the state X. For this case the two extra peaks corresponding to the state X are seen to decay with time when the samples are stored in the dark and in air, whereas those of the free base state are seen to grow. Two identical LB films of P1 deposited from a subphase with pH 2.70 were produced, one of which was stored in the dark and air, the other in the dark and under vacuum. Figure 7 shows the time dependence of the ratio of the absorption band of state X to that of the free base Soret band (PH2). After approximately 14 days (336 hours) the sample stored in the dark and air had returned almost entirely to the free base state, hence the ratio of peaks approaches zero. However, the sample stored under vacuum retains the peaks associated with the state X so that after 14 days the ratio stabilises at a value near 0.8. It thus seems that the state X is sensitive to either oxygen or water vapour or both.

Claims (6)

1. A sensor comprising a monomolecular layer of a porphyrin of formula:

wherein R is an aliphatic hydrocarbon radical containing a straight chain of at least 5 carbon atoms, or a plurality of such layers, a support for said layer or layers, means for controlling the degree of compression of the said layer or layers, and means for measuring variations of the optical absorption of said layer or layers in response to changes in the separation of the molecules of the said porphyrin in the said layer or layers brought about by adsorption into the said layer or layers of a substance to be detected.

2. A sensor according to claim 1 in which the said layer or layers is in contact with an aqueous phase.

3. A sensor according to claim 2 provided with means for controlling the pH of the said aqueous phase.

4. A sensor according to any one of the preceding claims in which R is a straight chain aliphatic radical of formula -CnH2 > 1, where n is 7 to 20.

5. A sensor according to claim 4 wherein R is -C8H17 or -C12H25.

6. A sensor according to any one of the preceding claims in which the means for measuring the optical absorption of the said layer or layers is a spectrophotometer able to measure absorption at wavelengths in the range 350 to 750 nm.