Fluorescent bead arrays by means of layer-by-layer polyelectrolyte ...

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Fluorescent bead arrays by means of layer-by-layer polyelectrolyte adsorption Andreas Schna¨ckel, Sabine Hiller, Uta Reibetanz and Edwin Donath* Received 1st September 2006, Accepted 10th October 2006 First published as an Advance Article on the web 2nd November 2006 DOI: 10.1039/b612117a Colloids with graduated fluorescence intensities were fabricated by means of layer-wise adsorption of fluorescein isothiocyanate-labelled poly(allyl amine hydrochloride) (FITC-PAH) together with poly(styrene sulfonate) (PSS) on silica particles. The graduated fluorescence was adjusted by variation of the fluorescent layer number and mixing labelled PAH with unlabelled PAH in one layer. The graduation of fluorescence intensities was adjusted in a geometric progression. It was shown that a proper label content is crucial if self-quenching phenomena are involved. The approach of mixing FITC-PAH with unlabelled polyelectrolyte during adsorption was unsatisfactory since competition in adsorption occurs. The system shows excellent stability at least over a period of two years.

Introduction Fluorescent colloidal particles are a powerful tool for a number of applications in life science and medicine. They can be qualitatively and quantitatively analysed by flow cytometry which is routinely applied in research, diagnostics and therapy.1–3 Immunological applications, such as suspension based immunoassays, require particles with well-defined and stable fluorescence which can be customized with specific immunoreagents on their surface. The colour and the species of the particle associated fluorophore respectively, is the coding parameter for the immunological property on the particle. It is essential that there is no interference of the particle fluorescence label with the epitope. The number of fluorescence channels in commercially available flow cytometric devices is limited. To simultaneously detect a sufficient number of analytes, cytokines or antibodies, for example, it is thus desirable to have well defined bead arrays with a sufficient number of fluorescence gradations.4,5 Cytometric bead array (CBA, BD Biosciences, San Jose, CA) and Luminex xMAP technology (formerly FlowMetrix) (Luminex Corporation, Austin, Texas) are commercially available systems used in research and diagnostic applications.3,4 Many analytes can be detected and quantified simultaneously in low sample volumes.6–11 Comparisons with ELISA demonstrate the advantages of these multiplex suspension arrays: they were found to be more accurate, sensitive and reproducible than the conventional ELISA procedure. Time and costs are comparable to, or less than, the ELISA.3–6 Currently, the fabrication of graduated fluorescence intensities for the different bead fractions is a relatively complex task involving covalent chemistry. Most often, the fluorescent dye is incorporated within the beads during their synthesis. Institute of Medical Physics and Biophysics, Leipzig University, Ha¨rtelstrasse 16-18, D-04107, Leipzig, Germany. E-mail: [email protected]; Fax: +49 (0)341 9715749; Tel: +049 (0)341 9715704

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The layer by layer (LbL) technique, however, may offer a very simple, and thus easily customizable, platform technique without much laboratory expense and equipment for custommade bead arrays. Different intensities in more than one colour can be combined and thus fluorescent bead libraries become possible. Another possibility of particle coding would be the scattering intensity. This can be combined with LbL. Copalis (coupled particle light scattering) multiplex technology (DiaSorin, Saluggia, Italy) uses scattering in flow cytometric multiplexing assays.3,12,13 Polyelectrolyte LbL deposition was introduced by Decher et al. in 1991. Thin multilayer films were assembled layer-wise onto a variety of surfaces by means of alternating deposition of polyanions and polycations.14,15 Later, the approach was transferred to colloidal particles.16 Silica, melamine formaldehyde and polystyrene latex particles, as well as biological cells have been used as substrates for film assembly.17,18 A large number of polymers and nanoparticles for the layer formation are available from chemistry and nature, e.g. proteins, carbohydrates, and nucleic acids.19–25 The layer-wise growth of the film allows for the fabrication of quite complex layers with inbuilt functions, e.g. fluorescently labelled polymers. The film structure can be controlled in a radial direction with nanometer precision.26 After film assembly the template core may be dissolved and removed so that a hollow capsule remains.27–29 Loading of capsules has been accomplished by various means. These capsules may be employed as microreactors, sensing devices or carriers.30,31 Drugs have been encapsulated and released afterwards.32 Fischlechner et al., 2006, introduced a novel surface display system by fusing a virus membrane with a lipid layer on top of the fluorescent LbL multilayer.20,21 It has been used as a multiplex suspension array for the detection and quantification of virus-specific antibodies.33 Fluorescently labelled layer constituents are an alternative to covalent chemistry-based particle labelling. Our principal This journal is ß The Royal Society of Chemistry 2007

objective is to show the possibilities and the limits of this LbL approach for fluorescence graduated particles. This is an important issue concerning possible applications in multiplex bead arrays. The challenge, however, is to produce a satisfactory large number of fluorescence gradation steps that can clearly be discriminated by their fluorescence and scattering properties in a standard flow cytometric analysis. Standard flow cytometric devices are equipped with two scattering channels and a limited number of fluorescence channels. Fluorescence intensities over 3–4 orders of magnitude can be resolved. It would be desirable to utilize the whole sensitivity range of each of the available fluorescence channels. This would require a large number of deposited layers to cover the available fluorescence intensity interval. On one hand, the number of adsorbed as well as of labelled layers is restricted. On the other hand, absorption, transfer and quenching phenomena as a result of the interaction between contiguous fluorescent layers have to be considered. Less precise layer formation and particle loss during the coating procedure become relevant with increasing layer numbers. To get a satisfying discrimination of fluorescence levels, the gradation steps have to be equidistant in log scale as well as wide enough to avoid overlapping. Also, the number of particle aggregates in the sample has to be minimized. Therefore, a study was undertaken to investigate the limitation of the technique with the aim of finding optimal conditions for fluorescence gradation by means of LbL. As an example, we selected the well established and reliable polymer couple for LbL formation, PAH as the polycation, combined with PSS as the polyanion. PAH/PSS forms stable multilayers, shows a linear layer growth in the radial direction and coated particles have a relatively low degree of aggregation.16 Fluorescent dyes, e.g. FITC or RITC (rhodamine isothiocyanate), can be easily bound to PAH.31,34 In combination with PAH/PSS we used 3 mm silica particles as the template because the size, being close to biological cells, was optimal for flow cytometry. FITC was chosen as the model dye. It is an inexpensive fluorescent dye, which can be easily bound to PAH. It shows a number of phenomena relevant for the fluorescence gradation design, for example self-quenching35 and transfer which would lead to pronounced interaction given the close distances between layers.26 Hence, there is the issue of selecting the appropriate label content to ensure maximum fluorescence since, generally, a high fluorescence yield would be advantageous for a high resolution of intensity levels.

Experimental Materials Silica particles, 3.09 mm in diameter (¡0.25 mm), were purchased from microparticles GmbH (Berlin, Germany) as a 5% (w/w) suspension. PAH (Mw: 70 000 g mol21; #750 monomers), PSS (Mw: 70 000 g mol21; #340 monomers), dextran sulfate sodium salt (DXS, Mw . 500 000 g mol21) and FITC (Isomer 1) are chemicals from Sigma (Deisenhofen, Germany). Sephadex G-25 chromatography gel was purchased from Sigma. Spectra/Por molecular porous membrane tubing This journal is ß The Royal Society of Chemistry 2007

(MWCO: 12000–14000 Da) is a product from Spectrum Laboratories, Inc. (California, USA). All other standard chemicals were obtained from Fluka (Neu-Ulm, Germany). Labelling of PAH FITC was covalently bound to PAH in a 3 : 1 methanol–water mixture. The remaining FITC was firstly removed by dialysis in 0.1 M NH4OH–NH4Cl buffer solution, pH 8.2. The NH4OH–NH4Cl buffer acts as a stop reagent. As a second step remaining FITC traces were removed by gel chromatography in a sephadex G 25 column. Covalent binding of RITC to PAH was conducted as follows: PAH was solved in 0.1 M carbonate buffer, pH 8.5, and RITC, dissolved in methanol, was added dropwise. After 2 d of incubation remaining free RITC was separated from RITC-PAH by dialysis and extraction with butanol. Purified FITC-PAH and RITC-PAH were lyophilized and stored at 4 uC. The degree of labelling was determined with a UV/vis spectrometer Cary 50 (Varian Instruments, USA) using the molar extinction coefficients e490 = 80 900 M21 cm21 for FITC-PAH and e559 = 62 100 M21 cm21 for RITC-PAH. Coating of silica particles Particles were coated at neutral pH in 0.1 M NaCl. 1 ml of polyelectrolyte solution (1 mg ml21) was used for coating. 500 ml of the particle suspension (5% by weight) were pipetted into the polyelectrolyte solution to a final volume of 1.5 ml and incubated under slight shaking for 10 min. Afterwards the samples were centrifuged at 650 6 g for 2 min and washed three times with 2 ml of distilled water after removal of the supernatant. In case of PAH being the top layer a 0.07 M NaCl solution was used for washing. Characterization with flow cytometry Fluorescence and scattering were recorded with a FACS Calibur flow cytometer (Becton Dickinson, USA) equipped with a 488 nm argon laser for excitation. The intensity interval that is detected by the fluorescence and scattering channels can be adapted to the samples by changing the detector amplification of each channel: The voltage reading for the FL-1 channel (FITC channel) was constantly adjusted at 630. In every measurement at least 10 000, in most cases 20 000 events, were recorded. The recorded data was analysed with the FACS software winmdi 2.8.

Results and discussion The functionalization of colloidal particles by coating them stepwise with polyelectrolyte layers is a rather natural way for the fabrication of particles with gradated fluorescence intensities. Indeed, varying the number of fluorescent layers a fluorescence gradation can be achieved. Flow cytometers show the best resolution in log scale. This is partly due to the log normal distribution of particles. Hence the fluorescence intensity was adjusted in a geometric progression with intensity steps as 8, 4, 2, 1, 1/2, 1/4…1/64. This is shown in Scheme 1. The deposition protocol is provided in Table 1. An arithmetic progression would give a strong overlapping of the recorded Soft Matter, 2007, 3, 200–206 | 201

Specifics of LbL

Scheme 1 PAH/PSS multilayer coated silica particle. Silica particles, 3 mm in diameter, were used for fluorescence graduation employing the layer by layer method. The layer deposition protocol is given in Table 1.

The number of fluorescent layers which can be assembled in a reasonable way by LbL is limited for various reasons. First, it is restricted because of increasing interaction and quenching between contiguous fluorescent layers. Even more important, there is some loss of control of layer growth with increasing layer number. Also, more and more particles are lost during the repeated coating and washing procedures. Another issue is aggregation of particles during layer built up. The degree of aggregation depends on the nature of polyelectrolytes and on the deposition and washing conditions. Particles coated with the couple PAH/PSS show a small tendency to aggregate. Aggregates of different order, i.e. single particles, doublets, triplets, quadruplets, etc. can be identified by their stepwise increased forward scattering and fluorescence intensity (Fig. 1). A high percentage of aggregates would be a serious handicap for the fluorescence resolution. Doublets would have the same fluorescence as the next higher in fluorescence intensity particle population, as it follows from the 2n gradation. In principle, gating on the basis of light scattering would help to overcome this particular problem, but given the width of the distribution it is always advantageous to avoid aggregation as much as possible. In this aspect the PAH/PSS couple is rather suitable, even though a relatively small number of aggregates remain. The importance of aggregation is illustrated in Fig. 1. Instead of PSS we assembled dextran sulfate as the top layer

distributions. The geometric progression approach ensures optimal spacing and thus fits nicely to the logarithmic scale of the FACS device. Table 1 Layer deposition protocol for fluorescence graduation by LbL. Note, that although the number of fluorescent layers varied, the total number of polyelectrolyte layers was kept constant at 18 Sample namea and proportion of labelled FITC-PAH in the coating solution

Layer Polyelectrolyte number species 1/64 1/32 1/16 1/8 1/4 1/2 1 2 4 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

PAH PSS PAH PSS PAH PSS PAH PSS PAH PSS PAH PSS PAH PSS PAH PSS PAH PSS

1/64 1/32 1/16 1/8 1/4 1/2 1 1 1 1

a

1 1 1 1 1 1 1 1 1 1 1

In the case of 1/64–1/2 the sample name refers to the proportion of labelled FITC-PAH within the 3rd layer; in the case of 1–8 the sample name refers to the total number of FITC labelled layers.

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Fig. 1 Histograms of flow cytometric measurements of silica particles, 3 mm in diameter, coated with (PAH/PSS)4PAH/DXS (A) and with (PAH/PSS)5 (B). The 3rd and 7th layers contain FITC labelled PAH. M1 denote singlets, M2 doublets, M3 triplets and M4 quadruplets of particles. (C) Mean fluorescence intensity as a function of the aggregate fraction corresponding to (A).

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(1A) and compared the aggregation with that of PSS as the top layer (1B). In both samples the 3rd and the 7th layer consisting of PAH were labelled with FITC. By comparing graphs A and B in Fig. 1, it is evident that the percentage of single particles, marked as the M1 region, differs considerably. In the case of dextran sulfate as the top layer (graph A) the M1 region is only 36% of the total count, and if PSS was the top layer (graph B) the M1 region covers 78% of the counted events. A similar behavior can be found in the forward scattering channel. Furthermore, a linear correlation between aggregate size and fluorescence intensity can be observed. This behavior is shown in Fig. 1C. It has to be mentioned, that with the couple PSS/ PAH, a considerable part of the recorded aggregates become dissociated upon the addition of the next polyelectrolyte species, thus ensuring a limited growth of aggregation upon ongoing layer assembly. Quenching A series of experiments with varying degrees of polyelectrolyte labelling was conducted. The aim was to find out a label ratio that provided maximum fluorescence. In each sample the 5th layer consisted of FITC-PAH. Fig. 2 shows the mean fluorescence intensities of the single particle fraction as a function of the degree of labelling. From 0.13 label per 100 monomers up to 0.6 label per 100 monomers there is an increase in the fluorescence intensity per particle. From 0.75 FITC per 100 monomers to 9.3 FITC per 100 monomers the fluorescence decreases. Two opposite trends are superimposed: an increase of the intensity with increasing content of dye molecules is met by a decrease caused by self-quenching, becoming more and more significant with increasing label concentration. Self-quenching, being responsible for the presence of the maximum in Fig. 2, becomes important at label ratios well below 1 : 100. Graduated fluorescence intensities Next, the fluorescence intensity was measured as a function of the number of labelled layers. The deposition protocol involves fluorescent layer ‘‘numbers’’ below one, such as ‘‘1/2, 1/4…1/64’’. The meaning of this notation is as follows: In

Fig. 2 Fluorescence intensities of particles coated with (PAH/PSS)3, the 5th layer is FITC-PAH.

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Fig. 3 Flow cytometric analysis of a mixture of fluorescence graduated silica particles. The label ratio was 0.75 FITC per 100 monomers in (A) and 0.13 FITC per 100 monomers in (B). Graphs (A) and (B) are fluorescence histograms of the gated R1 regions (singlets) in the respective dot plots (insets).

‘‘1/2’’ the layer was adsorbed from a mixture of FITC labelled PAH and unlabelled PAH in a 1 : 1 ratio. Unexpectedly, only the samples ‘‘1/2’’ and ‘‘1/4’’ showed a detectable intensity resolved fluorescence. In samples ‘‘1/8, 1/16, 1/32 and 1/64’’ no stepwise resolved fluorescence was found, even not at a higher amplification. Interestingly, in sample ‘‘1/4’’ two fractions, one with fluorescence and one without, were distinguished. Next the samples ‘‘8, 4, 2, 1, 1/2, 1/4’’ were mixed to form a bead array. Two different label ratios were compared, 0.75 label per 100 monomers (3A) and 0.13 label per 100 monomers (3B), respectively. The resulting distributions of fluorescence intensities are shown in Fig. 3. The insets of Fig. 3 show dot plots of forward scattering (FSC-H) vs. fluorescence (FL1-H). In the R1 region, which represents the single particles, 6 fractions of different fluorescence intensities can be observed. Aggregates of two particles can be distinguished from singlets by a higher forward scattering and fluorescence intensity and have been marked as R2. Software assisted gating allows then to separately display the fluorescence arising from single particles. These data form the basis for the histograms in Fig. 3A and B. The six fractions in graph A can be clearly distinguished although there is some overlapping and the peak which refers to sample ‘‘1/4’’ is small. The distribution appearing around the origin of the fluorescence scale (region 1/4*) can be associated with the non-fluorescent fraction of sample ‘‘1/4’’. When sample ‘‘1/4’’ was measured alone, the sample is split Soft Matter, 2007, 3, 200–206 | 203

into two subpopulations, as displayed in Fig. 5. One fraction shows a fluorescence signal in the expected range of intensities while the other lacks fluorescence. In the second series of experiments the FITC-PAH label used for the fluorescence graduation was decreased to 0.13 FITC per 100 monomers. The intention was to fabricate particles showing fluorescence of a smaller intensity, since it was desirable to also have fluorescent particles covering the 101–102 intensity range of the FACS device. The results are shown in Fig. 3B. The fractions show almost no overlapping in their fluorescence intensities. Interestingly, sample ‘‘8’’ with the labelling ratio of 0.13/100 showed a higher fluorescence intensity than sample ‘‘8’’ in the former experiment, where a label ratio of 0.75/100 was investigated. The interval along the fluorescence scale which is covered by the various fluorescent particle populations is wider than for the previously investigated higher label content. In Fig. 2 the 0.13 label ratio showed only half of the fluorescence of the 0.75 label ratio when only one fluorescent layer was present. Yet, when all 8 layers of PAH were fluorescent, the lower label degree showed a comparable or even higher fluorescence intensity. This behavior can only be explained by the interaction of the FITC-labelled layers leading to an unfavourable decrease of the total fluorescence. Self-quenching, naturally important within one layer, obviously takes place between different layers as well. Its significance grows with the increasing number of labelled layers. Our conclusion was that it is more favourable to employ a label ratio as small as possible for a fluorescence graduation by the LbL method, at least in case of FITC. Imhof et al., 1998, investigated the fluorescence behavior of silica particles incorporating FITC at different ratios inside the colloidal spheres. It was concluded that colloids with low dye concentration are useful for photonic applications, referring to the increased energy transfer between the dye molecules as their concentration increases.35 On the other hand, care has to be taken that the label ratio is not too low. Otherwise it becomes impossible to form a homogenous (with regard to their fluorescence) particle population.

Fig. 4 Mean fluorescence intensity of single particles as a function of the number of labelled layers. Full squares correspond to a label ratio of 0.13 FITC per 100 monomers. Open circles represent a label ratio of 0.75 FITC per 100 monomers. The straight line is a theoretical prediction assuming fluorescence intensity is doubled in every step.

equivalent. Labelled PAH probably adsorbs at a smaller rate than the unlabelled molecule. The composition of the layer thus does not reflect the proportion in solution. Another exception are the samples ‘‘4 and 8’’ of the 0.75/100 label ratio experiment. Their fluorescence intensities do not reach the theoretically expected values. This is caused by the enhanced self-quenching with increasing number of labelled layers, as discussed above. The dot plots of the samples ‘‘1/16, 1/8, 1/4, 1/2’’ of the 0.75/100 label ratio are shown in Fig. 5. It demonstrates the breakdown of the concept of homogeneous labelling at low intensities caused by competitive adsorption. In every sample the singlets and doublets can clearly be distinguished from their scattering. A noticeable fluorescence

Competitive adsorption In Fig. 4 the mean fluorescence intensity for the two, 0.13/100 and 0.75/100, fluorescent particle gradations (peaks corresponding to Fig. 3) is given as a function of the number of labelled layers. The two straight lines represent the expected theoretical proportionality between labelled layers and fluorescence intensity in double log scale. Deviations from the theoretical expectations represent samples ‘‘1/4 and 1/2’’ of the 0.13/100 label ratio, where the fluorescence is significantly smaller than expected. This may be explained by different affinities of labelled polymer and unlabelled polymer to adsorb during the layer formation. FITC is negatively charged and thus slightly changes the total electric charge of the polyelectrolyte chain. It may form intramolecular bridges in PAH which affect the configuration of the molecule and may prevent the PAH from adsorbing completely flat to the PSS layer. Consequently the adsorption behavior of labelled and unlabelled PAH is not 204 | Soft Matter, 2007, 3, 200–206

Fig. 5 FACS dot plots as a function of the FITC-PAH fraction.

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was recorded only in samples ‘‘1/2 and 1/4’’. Samples ‘‘1/8 to 1/64’’ do not observe fluorescence (data for samples ‘‘1/32 and 1/64’’ not shown). In sample ‘‘1/4’’ two fractions with a different fluorescence behavior are distinguishable: one fraction which shows a fluorescence and another without fluorescence like the control. In sample ‘‘1/8’’ only very few particles with a fluorescence can be identified. The corresponding samples of the 0.13/100 label ratio behave similarly. It has to be mentioned that the label degree of 0.13/100 monomers employed in Fig. 3B, which gave the best resolution, refers to the lower limit for homogenous labelling. It corresponds to 1 FITC per molecule PAH (750 monomers). In principle, coupling of the FITC to the PAH is random. Thus, there are polymers without FITC and those with one and with more than one dye molecule. The labelling follows a Poisson distribution: 37% of the PAH molecules are actually not labelled, 37% have one FITC and the rest has two or more. Hence, it is quite unfavourable to mix labelled with unlabelled PAH for layer build up, if the fraction of ‘‘labelled’’ PAH becomes too small. For instance the coating solution of sample ‘‘1/2’’ of the 0.13/100 label contained only 18% of PAH, which carried one FITC molecule, in sample ‘‘1/4’’ only 9%. Considering this and the possible competitive behaviour of coating, the unsatisfactory results for the lower intensity range become more understandable. Fluorescence resonance energy transfer (FRET) An alternative approach to adjust low fluorescence intensities ranging between 101–102 would be to take advantage of FITCquenching by energy transfer to an acceptor dye. To this end, 75% FITC-PAH (0.4/100 monomers) were mixed with 25% RITC-PAH (0.93/100 monomers). This mixture was diluted with unlabelled PAH in a ratio 1 : 1 (referring to ‘‘1/2’’) and was used for the build up of one fluorescent layer within a multilayer system of (PAH/PSS)9. The particles then observed a fluorescence peak in the lower intensity range with a mean of 5 (Fig. 6). Fluorescence stability and storage life Another important issue concerning practical use is storage life and the stability of fluorescence over time. The samples were

Fig. 7 Fluorescence stability and storage life. The sample was measured at the beginning (2003), after one year (2004) and after 2 years (2005).

stored in the dark at 4 uC over a period of two years. Measurements were repeated after 1 year and two years, respectively, with identical settings of the flow cytometer. The histograms of the FITC-channel at the beginning, after one year and after two years of the 0.75/100 label ratio experiment are provided in Fig. 7. The results obtained after one year represented an almost exact match of the original recordings. After two years a slight decrease in the measured fluorescence intensity was observed. But the fractions were as distinguishable as before. The peaks were only shifted along the fluorescence scale. This can be explained by the replacement of the FACS machine. This proves the very good stability of the assembled system over time.

Conclusions The LbL approach was shown to be a simple and suitable method to fabricate a custom-made log-scale graduated fluorescence bead array. In the case of FITC self quenching had a considerable influence on the quality of the fluorescence gradation. Care has to be taken to adjust for a proper label/monomer ratio. A different adsorption behavior of labelled and unlabelled polyelectrolyte may limit a controlled graduation of fluorescence in the lower intensity region. The findings in this work, obtained with the model dye FITC, will certainly have relevance for other dye species employed in bead arrays.

Acknowledgements This work was supported by a grant from the German Ministry of Education and Science, BMBF 0312011C.

References Fig. 6 Particles with mixed FITC-PAH and RITC-PAH (3rd layer) for energy transfer. The histogram shows only singlets marked as R1 in the dot plot of the inset.

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