C. J.
McHugh
,
F. T.
Docherty
,
D.
Graham
* and
W. E.
Smith
Department. of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow, UK G1 1XL. E-mail: duncan.graham@strath.ac.uk; Fax: 0141 552 0876; Tel: 0141 548 4701
First published on 11th December 2003
The syntheses of a number of azo and azine dyes with various surface attachment groups is described. The dyes use different methods of achieving surface complexing and are evaluated for their suitability as multiple labels for SERRS. The surface complexing agents, 8-hydroxyquinoline, benzotriazole, and pyridine are both shown to form robust layers on the silver surface. The relative intensities of the SERRS signals from each dye were shown to be predictive by considering the molar absorption coefficient at the laser excitation frequency.
The introduction of a chromophore to a non-chromophoric analyte can be achieved by covalent attachment of functionalised dyes to create coloured molecules that can be easily detected. This can be done using extensive labelling chemistry already developed for techniques such as fluorescence, where the derivitisation of molecules containing the appropriate functional groups is relatively easy to achieve.7,8 For a dye to be an effective SERRS label it must be able to bind to the roughened metal surface and remain attached during the SERRS measurement. Although it is possible to adsorb the dye onto the surface by electrostatic attraction, the SERRS spectrum can be unreliable due to desorption, commonly observed below monolayer coverage, or orientational changes which can cause changes in intensity. Therefore for reproducible SERRS analysis and for coding nanoparticles the dyes must bind strongly to the silver surface. Here we report the syntheses of a number of new dyes for use as SERRS labels, which use different methods of surface complexing to form tightly bound layers on the surface and we evaluate their suitability for SERRS multiplexing.
The metal used for surface enhancement was a citrate reduced silver colloid, prepared by a modified Lee and Meisel method.10,11 For each sample 250 µL of silver colloid suspension, 250 µL of distilled water, 30 µL of analyte and 10 µL of 0.067 M spermine were mixed together and the SERRS signal was immediately acquired. Some of the dyes had limited solubility in water so dimethyl sulfoxide (DMSO) was used to make dye solutions of 1 × 10−3 M and they were diluted to lower concentrations using water. All spectra have been normalised so that the most intense feature in the spectrum has an intensity of one thousand counts. For each dye, five consecutive spectra were acquired to check the spectra were reproducible. With the exception of dye 4, which has a poor signal to noise ratio, the peak positions and relative intensities were reproducible for all of the dyes.
Dye 2, 1-[4-(8-hydroxy-quinolin-5-ylazo)-phenyl]-ethanone was synthesised to investigate the use of alternative surface complexing groups. This dye has the same keto function for attachment to the analyte molecule, but the benzotriazole surface attachment group has been replaced by 8-hydroxyquinoline. Of the seven possible hydroxyquinolines, only 8-hydroxyquinoline forms chelate compounds with metals.17 The use of these chelate compounds for the detection of magnesium and aluminium is well known, but it has also been used to form chelates with silver.
Many of the dyes previously synthesised for use specifically as SERRS labels are azo dyes. In order to investigate the effect of the azo chromophore, an azine linkage has been used in place of the azo. Dye 3, N,N′-bis-quinolin-8-ol-5-ylmethylenehydrazine has the same metal attachment group as dye 2 to allow the effect of the azine group to be compared with the azo. The azo–azine comparison was further investigated by comparing dyes 4, N,N′-bis-pyridin-3-ylmethylenehydrazine and 5, 4,4′-azopyridine. Both of these complex to the metal surface through pyridine. The use of pyridine to complex with silver is synonomous with the first SERS experiments, being the first molecule to exhibit surface-enhanced Raman scattering through adsorption at the metal surface.18 Here, both ends of the molecule are expected to complex to silver, thus forming a polymeric layer on the surface. Crystal structures of two silver complexes obtained from dyes 4 and 5 indicate that this is correct.19 This is expected to provide stability to the surface species.
Fig. 2 shows the spectra acquired from each of the individual dyes. It can be seen from Fig. 2 that good SERRS signals are obtained from all of the dyes except dye 4. Some common features are observed in dyes that contain the same functional group. For example, the peaks observed between 1420 and 1470 cm−1 are due to azo stretches, and the peaks around 1600 cm−1 can be assigned to phenyl ring quadrant stretches. The intensities of these features compared to other features in the spectrum vary for the different dyes. This means that changes in relative intensity, as well as changes in peak position can be used to distinguish between different components in multiplex analysis using SERRS.
One area of particular interest for SERRS multiplexing is the detection of target DNA sequences which are each labelled with a unique SERRS dye. The correlation of an individual dye to a particular DNA sequence will allow detection of a number of disease states simultaneously. Therefore, to test the suitability of these dyes for multiplex analysis an attempt was made to simultaneously detect a commercially available dye-labelled oligonucleotide and dyes 1, 2 and 3. The oligonucleotide examined contained a basic priming sequence of 5′ GTG CTG CAG GTG TAA ACT TGT ACC AG 3′. The visible chromophore used was the fluorophore 2,5,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein (HEX), which was attached at the 5′ terminus. HEX is negatively charged and therefore repels the negatively charged metal surface. Surface attachment was achieved by the incorporation of positively charged modified nucleobases at the 5′-terminus next to the HEX label.20 Spermine was again used as the aggregating agent since it is known to neutralise the negatively charged phosphate backbone of the oligonucletide,21 therefore removing any repulsion between the analyte and metal surface.
To investigate the effect of using mixtures of dyes to create specific coding on the particle surfaces a similar SERRS experiment to that used for single dyes but using 30 µL of each dye solution was carried out. To ease the interpretation of the spectrum from all four analytes it is desirable that the peaks from each of the dyes have similar intensities. When the spectra from the individual dyes were acquired at the same concentration there were large variations in the intensities of the signals collected from each dye, even though the same acquisition time was always used. This suggested that the dyes had to be used in different concentrations to have similar intensities in the multiplex spectrum. The optimal concentrations were obtained by optimising the concentration in a two-dye mixture to obtain the desired intensities, then repeating the experiment to incorporate a third dye, and finally the fourth.
Fig. 3 shows the spectra recorded from the dye mixture and from the individual dyes at the concentrations used in the mixture. All spectra have been normalised to have the same maximum intensity in the highest peak in each spectra. In this figure dashed lines have been added linking features in the spectrum from the mixture with the spectrum from the pure sample from which they originate. Table 1 summarises the marker bands for each dye in the spectrum from the mixture and states the volumes and concentrations of the dyes used in the mixture.
Sample | Concentration/mol L−1 | Volume/µL | Marker bands |
---|---|---|---|
HEX | 1 × 10−7 | 30 | D, G, G*, J |
Dye 1 | 1 × 10−5 | 30 | I |
Dye 2 | 1 × 10−5 | 30 | A, B, C, D, E*, F |
Dye 3 | 1 × 10−4 | 30 | E, G, H |
It is clear that the peaks labelled A, B and C are due to the presence of dye 2. Peak D has contributions from both dye 2 and HEX. The relative intensity of this peak is higher in the spectrum from the mixture than in either of the individual spectra from HEX or dye 2 showing that the presence of both samples have contributed to this feature. A shoulder, E*, is visible on peak E. It is likely that E and E* originate from contributions from dye 3 and dye 2 respectively, which have peaks at similar positions. Feature F can be attributed to dye 2 and the peak G, and its shoulder G* look similar to the feature in HEX at that wavenumber. However, dye 3 also has a peak at the position of peak G. The relative intensity of G* compared to G has decreased in the spectrum from the mixture compared to that of pure HEX, indicating that the contribution from dye 3 has increased the intensity of peak G in the mixture. In the pure HEX spectrum peak G is less intense than the peak at higher wavenumber. The difference in intensities between these peaks is not so large in the spectrum from the mixture, providing further evidence for a contribution from dye 3. Peak H is due to the presence of dye 3 and I can be attributed to dye 1. Dye 2 also has a peak at the position of peak I. However, examination of the spectrum from dye 2 shows that this peak is less intense than the group of peaks responsible for features A–C in that spectrum. That group of peaks has a low relative intensity in the spectrum from the mixture, and the peak at 1590 cm−1 will have an even smaller contribution to the spectrum from the mixture. Therefore peak I will be dominated by the contribution from dye 1 and the effect of dye 2 can be taken to be minor. Finally, peak J can be assigned to HEX.
Table 1 shows that a wide range of concentrations are required for the dyes to give spectral features of similar intensities. If dyes of this type are to be used for multiplexing it is essential that they are used at the correct concentrations, so that the absence of the SERRS signal from an analyte is due to it not being present, rather than the SERRS intensity from it being so small that it is swamped by the signals from other compounds. Therefore it would be useful to be able to predict the concentrations of the dyes required to generate SERRS marker bands of the correct intensity. Two factors which could affect this intensity are the strength of the dye adsorption onto the metal surface, and the absorption of the molecule at the laser excitation frequency. Both of these factors are now considered.
One aim of this work was to investigate the use of 8-hydroxyquinoline as the surface attachment group, compared to the more commonly used benzotriazole group. Dyes 1 and 2 have the same structure, apart from the different surface attachment group. Table 2 lists the wavelength of the absorption maxima and the molar absorption coefficient, ε,at this wavelength and at the laser excitation wavelength for all of the dyes studied. It is clear that both dyes have similar values of ε at the laser wavelength so any differences in their SERRS signals would be due to change in the surface complexing group. However, Table 1 shows that they are used in the same concentrations to get similar intensities in the dye mixture. This indicates that the 8-hydroxyquinoline group can be used instead of benzotriazole for surface attachment without lowering the intensities of the SERRS signals attained.
Dye | λ max/nm | ε/dm3 mol−1 cm−1 at λmax | ε/dm3 mol−1 cm−1 at 514.5 nm |
---|---|---|---|
1 | 442 | 44480 | 1632 |
2 | 403, 477 | 21624, 6772 | 1556 |
3 | 410 | 32820 | 1217 |
4 | 297 | 33010 | 0 |
5 | 460 | 26250 | 31 |
HEX | 535 | 73000 | 9882 |
The other 8-hydroxyquinoline containing dye is dye 3. In the dye mixture it has to be used at a higher concentration to give a similar SERRS intensity as dye 2. Since they have the same surface attachment group it is likely that differences in their absorption at the laser frequency is responsible for dye 3 having to be used at a higher concentration. This is confirmed in Table 2 since the value of ε is around 20% lower for dye 3 than dye 2. The large effect on the SERRS signal of the value of ε at the excitation frequency is clearly demonstrated by considering HEX, which is used at the lowest concentration in the dye mixture and has a substantially larger value of ε at 514.5 nm than for any of the other dyes.
One major difference between dyes 2 and 3 is that one is an azo and one is an azine. However, they also have other structural differences so a better understanding of the effect of the azo/azine group on ε can be gained by comparing dyes 4 and 5, which are structurally more similar than dyes 2 and 3. Both complex to the silver through pyridine to form linear polymers, so surface attachment is not the reason for the poor spectrum from dye 4 since a good signal to noise ratio is obtained from dye 5. Table 2 shows that dye 5 only absorbs very weakly at the laser frequency and no signal could be detected from dye 4 which would explain the poor SERRS signal from it. This provides further evidence that absorption from an azine is not as good as from an azo at this excitation frequency and that the value of ε at the laser frequency is a good indicator of the magnitude of the SERRS signal.
Footnote |
† For Part 1 see ref. 12 |
This journal is © The Royal Society of Chemistry 2004 |