Journal of Chromatography A, 1156 (2007) 154159

Miniaturised isotachophoresis of DNA

Jeff E. Prest a, , Sara J. Baldock a , Philip J.R. Day b , Peter R. Fielden a , Nicholas J. Goddard a , Bernard J. Treves Brown a
School of Chemical Engineering and Analytical Science, The University of Manchester, P.O. Box 88, Manchester M60 1QD, UK b CIGMR, The University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK Available online 12 January 2007
a

Abstract This paper presents the ndings of a feasibility study investigating the behaviour of DNA under conditions of miniaturised isotachophoresis. An electrolyte system comprising a leading electrolyte of 5 mM perchloric acid at pH 6.0 and a terminating electrolyte of 10 mM gallic acid was devised and used to perform isotachophoresis of DNA containing samples on a miniaturised poly(methyl methacrylate) device. Under such conditions it was found that no separation of DNA fragments was observed with the substance migrating instead as a single isotachophoretic zone. Whilst such a result shows the method is unsuitable for analysis DNA it offers signicant potential as a means of sample preparation for subsequent analysis using another method. This is because the single zone of DNA formed is preconcentrated to a constant concentration governed by the leading ion and is separated from all species with different effective electrophoretic mobilities. 2007 Elsevier B.V. All rights reserved.
Keywords: DNA; Isotachophoresis; Sample preparation; Miniaturisation; Microdevice; Chip

1. Introduction Electrophoretic methods have proven themselves to be useful tools in analysing nucleic acids. In particular, the use of capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE) have been found to be effective methods for the separation of DNA fragments [1,2]. These methods have also proven readily amenable to implementation on a miniaturised scale [3,4]. Miniaturisation offers a range of benets including improved analytical performance, reduced analysis times and the relative ease of performing parallel analyses. These latter two features allow for high throughput operations and were a major part of the reason why these electrophoretic methods were widely used in the project to map the human genome [5]. Isotachophoresis (ITP) is another member of the family of electroseparation techniques, albeit one that is somewhat less commonly encountered than CZE. ITP does however offer a number of useful features not found in CZE. The most useful of these is the ability to control separation parameters by vary-

Corresponding author. Tel.: +44 161 306 8900; fax: +44 161 306 4896. E-mail address: j.prest@manchester.ac.uk (J.E. Prest).

ing the electrolyte system used. The concentration of the leading electrolyte governs the concentration that all of the sample zones adopt, enabling ITP to be used as a method to preconcentrate dilute samples. Like many other electroseparation methods ITP can be readily miniaturised. When performed in such a format the technique has been used as both a separation method in its own right [6,7] or as a sample preparation step prior to performing a CZE separation [8,9]. Miniaturised ITP has been used with a wide variety of samples as evidenced by the range of applications shown in a recent review on the topic [10]. However, to date there have been few reported uses of miniaturised ITP involving DNA samples. The use of the method has seen some use as a preconcentration method applied either conventionally (ITPZE) [11] or as transient ITP (TrITPCGE) [12,13] for separations of DNA fragments. However, none of these reports investigated in detail the behaviour of the ITP stage. This paper reports the rst known investigation into the behaviour of DNA under conditions of miniaturised isotachophoresis. As part of the study an electrolyte system was devised which enabled DNA to be isolated and migrated isotachophoretically on a poly(methyl methacrylate) (PMMA) chip device. The developed system was subsequently tested with samples of salmon sperm DNA and human genomic DNA extracted from whole blood.

0021-9673/$ see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.01.026

J.E. Prest et al. / J. Chromatogr. A 1156 (2007) 154159

155

2. Experimental 2.1. Instrumentation The miniaturised PMMA separation device used in this work was fabricated using a previously described direct milling procedure [14]. Sealing of the device was achieved by means of a piece of self-adhesive, 400 m thick, polyester laminate (Ritrama, Monza, Italy). The device comprised of two main channels, one linking the cross to the junction point, which had a length of 57 mm, and the second running between the junction point and well B. This latter channel was 200 m wide and 300 m deep and incorporated a conductivity detector with on-column 75 m diameter platinum wire electrodes (Aldrich, Gillingham, Dorset, UK) arranged in an opposed conguration. The detector was located at a distance of 44 mm from the junction point. All other channels were 300 m wide and 300 m deep. A schematic diagram of the channel network is shown in Fig. 1. The overall size of the device was 78 mm wide by 78 mm long. A PS350 high voltage, 5 kV power supply (Stanford Research Systems, Sunnyvale, CA, USA), congured to supply negative voltages was used to provide the constant currents required to drive the separations. Conductivity detection was achieved using a system built in-house, which uses capacitive coupling to isolate the low voltage detection circuitry from the high separation voltages. Electrolyte and sample loading was performed using a gravity feed hydrodynamic uid transport system detailed in an earlier paper [14]. However, a change was made to the sample reservoir in this study. To reduce the volume of sample required a 1000 l disposable pipette tip was used for this purpose in place of a barrel of a 2.5 ml disposable syringe. Control of the power supply, uid transport system and data acquisition from the detector was carried out using a standard PC with programs written in-house using LabVIEW software (version 7.1, National Instruments, Austin, TX, USA). Full details of the instrumentation have been previously described by the authors [15].

Table 1 Separation program used to carry out miniaturised isotachophoretic separations Step Time (s) Current ( A) Valve status A 1 2 3 4 5 6 7 20 20 1 0.5 0.5 80 1000 0 0 0 0 0 25 10 x x x o o x x B o o x x x x x C x x o x x x x D x o o o x x x E o x x x o x x

Here, x: closed; o: open.

2.2. Separation Conditions A seven step control program, shown in Table 1, was used to perform all of the separations carried out during this investigation. The system is ushed and the device is loaded with leading electrolyte in steps 1 and 2. Terminating electrolyte is then loaded in step 3. Steps 4 and 5 inject sample into the device. The former step positions the sample at the cross whereas the latter lls the separation channel between the cross and junction point, thus giving an injection volume of 5.1 l. The actual isotachophoretic separation begins in step 6 with a current of 25 A applied between wells B (ground) and C. In step 7, the applied current is reduced to 10 A, to slow down the separated zones for detection purposes. 2.3. Chemicals The compositions of the electrolytes used in this work are given Table 2. The leading electrolyte was produced using perchloric acid (70%, Riedel-de Ha n, Gillingham, Dorset, UK) e with Mowiol (40-88, Aldrich, Gillingham, Dorset, UK) added to suppress electroosmotic ow. The pH of the leading electrolyte was adjusted using 2-methylbenzimidazole (98%, Fluka, Gillingham, Dorset, UK). The terminating electrolyte was gallic acid monohydrate (>99%, Acros, Loughborough, UK). Model samples were prepared using low molecular weight salmon sperm DNA (Fluka). Samples and electrolytes were prepared using >18 M water (Elga Maxima Ultra Pure, Vivendi Water Systems, High Wycombe, UK).

Table 2 Composition of the electrolyte system developed to allow the isotachophoretic migration of DNA Electrolyte Ion Concentration (mM) pH buffer pH Additive Concentration (g l1 ) MBI: 2-methylbenzimidazole. Leading Perchlorate 5 MBI 6.0 Mowiol 0.5 Terminating Gallate 10

Fig. 1. Schematic diagram of the channel network in the miniaturised PMMA separation device. All channels are 300 m wide and 300 m deep with the exception of that running from well B to the junction point which is 200 m wide and 300 m deep. Letters A, B, C, D and E refer to the wells into which the inlet/outlet connections to the device are made. Sample enters through well A, leading electrolyte through B and terminating electrolyte through C. Wells D and E exit to waste.

156

J.E. Prest et al. / J. Chromatogr. A 1156 (2007) 154159

3. Results and discussion 3.1. Design of Electrolyte System Previously there have been no reported studies into the behaviour of DNA under conditions of miniaturised ITP. Therefore, it was necessary to develop a new electrolyte system for use in this study. As it was not known what kind of separation, if any, would occur when such a procedure was attempted it was thought useful to develop an electrolyte system suitable for use in sample preparation situations. In such circumstances, the primary demand on an electrolyte system is to allow a pure zone of the particular species of interest to be produced, rather than allowing the simultaneous separation and determination of a wide range of species. One of the useful features of isotachophoresis is the ability to tailor the electrolyte system to eliminate interference from other species present in the sample matrix [16]. This can be done by restricting the number of species in a sample which migrate isotachophoretically by using a leading electrolyte and terminating electrolyte with only a relatively small mobility difference between them. The ndings of a previous study using free solution ITP as a preconcentration method for a CGE separation indicated that DNA exhibits a mobility a little higher than that of acetate at pH 8.3 [17]. Therefore, acetate could be a possible terminating ion. However, it was thought prudent to use a slightly slower species, gallate, in case DNA exhibited a slower than expected mobility under conditions of miniaturised ITP. The leading ion selected for this study was perchlorate. This species has a lower mobility than the ubiquitous inorganic anions chloride, nitrate and sulphate. Thus, these species should not cause any interference. DNA exhibits a constant charge-to-size ratio between pH 6.0 and 8.0 [18]. This means that the effective mobility over this range should be constant and similar to that at pH 8.3 referred to above. For this work, a leading electrolyte pH of 6.0 was used. This pH was selected to minimise potential interference from carbonate which arises from dissolved atmospheric carbon dioxide. A low concentration (5 mM) leading ion was decided upon to hopefully negate any possible precipitation problems. Full details of the electrolyte system can be found in Table 2. 3.2. Separations Prior to performing any experiments in this study it was not known what the outcome would be when samples of DNA were subjected to miniaturised ITP. This was because it had been previously suggested that the use of capillary scale ITP was not the ideal method for the separation of oligonucleotides (and by inference DNA) [19]. However, gel based ITP had previously been shown to offer some potential for the separation of DNA fragments [20]. Therefore, initially it was necessary to perform a series of experiments to identify what kind of separation arose. These preliminary experiments were carried out using low molecular weight salmon sperm DNA (<2000 bp) to produce samples. Using the devised electrolyte system it proved possible to get the DNA migrating isotachophoretically between the leading and terminating electrolytes. An example of such a

Fig. 2. Isotachopherogram produced when a sample containing 125 g ml1 salmon sperm DNA was analysed using miniaturised ITP. Leading electrolyte 5 mM HClO4 , 0.5 g l1 Mowiol, pH 6.0 (2-methylbenzimidazole). Terminating electrolyte 10 mM gallic acid. LE: leading electrolyte; TE: terminating electrolyte.

result, for a sample containing 125 g ml1 salmon sperm DNA, is shown in Fig. 2. From the illustrated result it can be see that the DNA produced a sharp zone when subjected to miniaturised ITP. This result is somewhat different to that observed in a previous report of using capillary scale ITP to analyse nucleic acids, including DNA [21]. In this previous study, the aim was to separate DNA fragments. To try and achieve this an ampholyte solution was added to the samples and this substance contributed to complexity of the observed results recorded using a potential gradient detector. This previous study used a leading electrolyte at pH 9.9 and also suffered some problems with carbonate interference. As mentioned above such interference was not a problem in the current work due to the electrolyte system developed. The results observed in the current study suggested that the DNA was migrating with a relatively homogenous effective mobility and that there was essentially no separation of the DNA into fragments. This result indicates the method was unlikely to be of use for separating DNA fragments. However, from a sample preparation perspective it was a very promising result as it indicated that all of the DNA was being concentrated into a single zone which could then be used in another procedure. Such an operation may be another separation process such as CZE or an amplication process such as a polymerase chain reaction (PCR). As ITP is a separation technique this zone of DNA will be separated from any other substances present in samples which have different effective mobilities. Such a feature is of signicant importance if the DNA is subsequently being subjected to PCR. This is because the amplication can suffer a loss of efciency by the presence of certain species such as heme, lipids or enzymes [22,23]. The isotachophoretic migration of DNA was found to occur in a reproducible manner. Results observed with samples of

J.E. Prest et al. / J. Chromatogr. A 1156 (2007) 154159

157

salmon sperm DNA showed a good level of repeatability. When ten consecutive runs were carried out using a sample containing 125 g ml1 DNA the observed relative step height (RSH) standard deviation (SD) was found to be 0.673 0.011. In this work, the RSH was taken as the ratio of the sample step height to the height of the terminating step. When conductivity detection is used in ITP the step height provides qualitative information about the substances being analysed. Good reproducibility was also witnessed in the quantitative information contained in the isotachopherograms, the zone lengths. The ten repeat runs of the 125 g ml1 salmon sperm DNA produced zones SD with a length of 19.8 0.42 s. The use of a low concentration, 5 mM, leading electrolyte allows for relatively fast analysis times. For example, the result shown in Fig. 2 was achieved in just over 3 min. This separation time included the 42 s to complete the injection program, so that the actual isotachophoretic separation was completed in under 160 s. Thus, it can be seen the miniaturised ITP is eminently suitable for use as a high throughput sample preparation method. To investigate the range of DNA concentrations over which the method could be used a series of nine samples were analysed. Four replicate runs were performed with each of the samples, which contained salmon sperm DNA concentrations ranging from 9.5 to 750 g ml1 . The results obtained from these experiments were used to check the linearity of the method by means of a calibration curve. It was found that good linearity was obtained with a correlation coefcient of 0.998 calculated. The parameters of the curve, produced using weighted linear regression, were that the slope was 0.132 0.001 s ml g1 and the intercept was 2.94 0.37 s. The errors shown for both of these parameters represent the SDs. Using the regression equation a limit of detection (LOD) for salmon sperm DNA was calculated to be 8.4 g ml1 . This value was calculated using the intercept, which represents an estimation of the blank, plus three times the standard deviation associated with this parameter. The calculated LOD gives an indication of the concentration of DNA which is necessary to be present in a sample that is being pretreated so that it can be detected reliably using the conductivity detector used in this work. This gure is not necessarily the minimum concentration required to produce an isotachophoretic zone. Using an alternative detection method such as UV-absorbance may allow shorter zones to be detected. Such an effect can occur because UVabsorbance is a specic detection method and conductivity a universal detection method [24]. To realise the potential benets from such an approach would require an amendment of the electrolyte system used in this work as it would be necessary to change from using gallate to using a non-UV absorbing
Table 3 Results obtained with human genomic DNA samples Sample In-house 1 In-house 2 In-house 3 Tepnel 1
a

species as the terminating ion. The use of a specic detector such as UV-absorbance or uorescence (which offers the possibility of higher sensitivity [25]) may be useful if using ITP as a sample pretreatment method. This is because the species of interest can be bracketed by substances which are not detected by a specic detector. Whilst bracketing has been used in the past to improve quantication [26], the process can also be used to offer an accurate means of determining when to make the cut in the isotachophoretic stack to remove the required species. One of the useful features of ITP which makes it suitable for sample preparation purposes is the fact that the concentration of the zones in isotachophoretic stack are of a xed concentration. This concentration is governed by the choice of the electrolyte system and is approximately that of the leading ion. This feature means that for example the isolated DNA can be transferred for a subsequent operation such as PCR amplication on a volume added basis. This simplies the transfer operation as it eliminates the need to determine the concentration of the DNA. However, this same feature can make quantication of DNA using ITP somewhat problematic. As the zones formed are of a xed concentration, quantication can be achieved when using conductivity detection by measuring the length of the zone. The xed concentration of the zones means the higher the concentration present in the sample solution the longer the zone length produced. The problem with DNA samples is the polymeric nature of the substance. Thus, different samples can have variable molecular masses. Thus, although a calibration curve was produced for the salmon sperm DNA this calibration will not necessarily hold for other DNA samples. For example, the salmon sperm DNA used in this work has a mass of approximately 1.3 106 Da [27] whereas genomic human placenta DNA (Sigma, Gillingham, Dorset, UK) has a mass of approximately 10 106 Da. If DNA samples from a different source are analysed different zone lengths will be obtained for equivalent sample concentrations. This type of problem arises when any type of polymeric material is analysed using ITP. However, it was overcome to an extent when using ITP to analyse carboxymethylcellulose polymers by introducing an equivalents of carboxyl group factor into the calibration [28]. To implement this approach did, however, necessitate a prior analysis of the samples using a potentiometric titration. An alternative approach used for the isotachophoretic analysis of polyphosphates was to use an internal standard [29]. If quantication of DNA was required it may be possible to apply one of these approaches. Again the use of an alternative detection method may be benecial in reducing this problem.

Relative step height SD 0.629 0.686 0.675 0.632 0.039 0.076 0.020 0.019

Step length SD (s) 8.4 10.4 12.5 8.1 0.2 0.6 0.9 0.3

Sample volumea ( l) 50 50 60 45

Samples made up to 1000 l with deionised water prior to analysing with ITP standard deviations based on four replicates.

158

J.E. Prest et al. / J. Chromatogr. A 1156 (2007) 154159

To further investigate the behaviour of DNA when subjected to miniaturised ITP a series of analyses were made using a number of samples of genomic human DNA. The samples used were produced from whole human blood. These samples were puried by either an in-house method involving a phenol/chloroform extraction or using a commercial kit (Tepnel Life Sciences, Manchester, UK). Table 3 lists the RSHs and zone lengths observed with the genomic DNA samples. The volumes of the samples analysed are also given in this table. These values are different for the different samples as different amounts of the individual samples were available and the maximum possible volume was analysed. Fig. 3 shows an example of an isotachopherogram obtained with one of these samples (in-house 2). Generally, clear results exhibiting only a single obvious step were obtained with the genomic samples. However, with inhouse sample 1 an additional short zone with a RSH SD of 0.331 0.019 was noted. Longer analysis times were observed with all of the genomic samples compared to those of the salmon sperm samples. For example, the rear of the leading zone in the result shown in Fig. 3 with in-house sample 2 was detected after 227 s whereas in the example in Fig. 2 with the salmon sperm DNA the same feature was detected after 178 s. This nding indicates the presence of high mobility species in the genomic samples. Such species are likely to be inorganic salts such as chloride, nitrate or sulphate. These species all have higher effective mobilities than the perchlorate leading electrolyte. Thus, they will not produce an isotachophoretic step but will instead migrate within the leading electrolyte, resulting in a lengthening of this zone. As in ITP qualitative information is contained in the step heights produced, if what is detected with the genomic samples is DNA, there should be agreement between the RSHs observed with these samples and that of the salmon sperm DNA. To check whether this was the case the results were subjected to a Stu-

dents t-test. This statistical test was used to compare the results yielded by each of the genomic samples to those obtained with salmon sperm samples. The value used as the RSH SD for the salmon sperm DNA in these tests was taken as 0.652 0.023 (n = 36), which represents the results observed in producing the calibration curve. The results of the tests for all four genomic samples yielded t-values under the appropriate critical t-values at the 95% level. Thus, it could be said that statistically the steps observed were the same as with the salmon sperm samples and that what was being seen with the genomic samples was not signicantly different from salmon sperm DNA. Due to the earlier mentioned problems regarding calibrations, meaningful concentrations of the genomic samples could not be calculated. The differences in sample volumes analysed meant a simple comparison of zone lengths could not be used to determine the concentrations of DNA present in the samples compared to one another. However, it was thought that the calibration curve produced for the salmon sperm DNA could be used to enable a comparison of the concentrations of DNA present in the original genomic DNA samples to be made. When this was done it was found that in-house sample 3 had the highest concentration of DNA followed by in-house sample 2 and in-house sample 1 had the lowest concentration of DNA. This result was in agreement with the concentrations of the samples determined using a ND-1000 UV spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), which were that in-house sample 1 contained 113 g ml1 of DNA, sample 2 contained 202 g ml1 of DNA and sample 3 contained 334 g ml1 of DNA. These results indicate that the DNA from the genomic samples was successfully separated from the samples and concentrated up into isotachophoretic zones. The DNA concentration of the sample extracted using a commercial kit was found to be 350 g ml1 using the NanoDrop instrument. However, the results obtained using miniaturised ITP suggested the concentration was between those of in-house samples 1 and 2. It is not known whether this discrepancy was related to the extraction method used or due to the DNA in this sample having a different mass. 4. Conclusions When DNA samples were subjected to miniaturised ITP separations they were found to form an essentially homogenous zone with little evidence of fragmentation. Whilst such a result is of little use from the point of view of analysing the DNA it offers potential as a sample preparation method. This is because the zone of DNA formed has a xed concentration governed by the electrolyte system. This means that the DNA zone can be transferred to a subsequent further operation on a volume basis and also allows for preconcentration of dilute samples. This advantage is seen as being particularly benecial for preparing samples for PCR amplication. The separation mechanism of ITP allows for the elimination of unwanted matrix components to be achieved. In the current study, such an effect was achieved with genomic DNA samples extracted from whole human blood but it should be possible to use the method to for example isolate DNA from cell lysis products.

Fig. 3. Analysis of a sample of genomic human DNA from whole blood analysed using miniaturised ITP. Conditions used as given in Fig. 2.

J.E. Prest et al. / J. Chromatogr. A 1156 (2007) 154159

159

The use of miniaturisation technology allows for the procedure to be carried out rapidly. With the device used in this work samples produced using salmon sperm DNA were separated in under 200 s whereas those containing genomic DNA were separated in under 280 s. Thus, the method has potential for high throughput sample preparation. Indeed, if required these gures could be improved upon further with a different design of device. The times indicated also include the preparation steps of loading the device. This part of the process can be achieved rapidly as the separations are performed in free solutions rather than gels. The procedure is also easy to implement requiring relatively simple instrumentation. Acknowledgement This work was funded by the Medical Research Council (UK). References
[1] D.A. McGregor, E.S. Yeung, J. Chromatogr. A 652 (1993) 67. [2] C. Sumita, Y. Baba, K. Hide, N. Ishimaru, K. Samata, A. Tanaka, M. Tsuhako, J. Chromatogr. A 661 (1994) 297. [3] L.C. Waters, S.C. Jacobson, N. Kroutchinina, J. Khandurina, R.S. Foote, J.M. Ramsey, Anal. Chem. 70 (1998) 158. [4] D.-K. Kim, S.H. Kang, J. Chromatogr. A 1064 (2005) 121. [5] J.C. Venter, M.D. Adams, E.W. Myers, P.W. Li, R.J. Mural, G.G. Sutton, H.O. Smith, M. Yandell, C.A. Evans, R.A. Holt, et al., Science 291 (2001) 1304. [6] M. Mas r, D. Kaniansky, R. Bodor, M. J hnck, B. Stanislawski, J. Chroa o matogr. A 916 (2001) 167. [7] J.E. Prest, S.J. Baldock, P.R. Fielden, N.J. Goddard, B.J. Treves Brown, Analyst 130 (2005) 1375.

[8] R. Bodor, V. Madajov , D. Kaniansky, M. Mas r, M. J hnck, B. Stanisa a o lawski, J. Chromatogr. A 916 (2001) 155. [9] R. Bodor, D. Kaniansky, M. Mas r, K. Silleov , B. Stanislawski, Eleca a trophoresis 23 (2002) 3630. [10] L. Chen, J.E. Prest, P.R. Fielden, N.J. Goddard, A. Manz, P.J.R. Day, Lab Chip 6 (2006) 474. [11] A. Wainright, U.T. Nguyen, T. Bjornson, T.D. Boone, Electrophoresis 24 (2003) 3784. [12] Z.Q. Xu, T. Hirokawa, T. Nishine, A. Arai, J. Chromatogr. A 990 (2003) 53. [13] Z. Xu, T. Nishine, A. Arai, T. Hirokawa, Electrophoresis 25 (2004) 3875. [14] J.E. Prest, S.J. Baldock, P.R. Fielden, N.J. Goddard, B.J. Treves Brown, Analyst 127 (2002) 1413. [15] J.E. Prest, S.J. Baldock, P.R. Fielden, N.J. Goddard, K. Kalimeri, B.J. Treves Brown, M. Zgraggen, J. Chromatogr. A 1047 (2004) 289. [16] J.E. Prest, S.J. Baldock, P.R. Fielden, N.J. Goddard, B.J. Treves Brown, Analyst 128 (2003) 1131. [17] M.J. van der Schans, J.L. Beckers, M.C. Molling, F.M. Everaerts, J. Chromatogr. A 717 (1995) 139. [18] S. Auriola, I. J askel inen, M. Regina, A. Urtti, Anal. Chem. 68 (1996) a a 3907. [19] G. Bruchelt, D. Niethammer, K.H. Schmidt, J. Chromatogr. 618 (1993) 57. [20] M.P. Bellini, K.L. Manchester, Anal. Biochem. 268 (1999) 21. [21] H. Yamamoto, T. Manabe, T. Okuyama, J. Chromatogr. 480 (1989) 331. [22] M. Kubista, J.M. Andrade, M. Bengtsson, A. Forootan, J. Jon k, K. Lind, a R. Sindelka, R. Sj back, B. Sj green, L. Str mbom, A. St hlberg, N. Zoric, o o o a Mol. Aspects Med. 27 (2006) 95. [23] P.-A. Auroux, Y. Koc, A. de Mello, A. Manz, P.J.R. Day, Lab Chip 4 (2004) 534. [24] M. Svoboda, J. Vack, J. Chromatogr. 119 (1976) 539. [25] J.C. Reijenga, T.P.E.M. Verheggen, F. Everaerts, J. Chromatogr. 283 (1984) 99. [26] G. Eriksson, Anal. Biochem. 109 (1980). [27] K. Tanaka, Y. Okahata, J. Am. Chem. Soc. 118 (1996) 10679. [28] L.R. Whitlock, J. Chromatogr. 363 (1986) 267. [29] I. Motooka, K. Nakazaki, H. Nariai, M. Tsuhako, J. Chromatogr. 367 (1986) 271.