The most widely used substrate for DNA microarrays is glass whose chemical modification is well established (12
). However, well developed chemistries on polymeric surfaces are still in their infancy. So far, PMMA has been modified by Bulmus et al
) and by Henry et al
) to yield aminated PMMA sheets. In the present work, we have developed a surface chemistry that yields primary amines on a PMMA surface, and compared with the previously described chemical methods is both simpler and faster (Fig. A and B).
A fluorescent-based technique was used to quantify the number of NH2
groups on PMMA slides after chemical modification. FITC is a fluorescent dye that is specifically reactive towards primary amines (30
). The NH2
surface density was estimated from a standard curve (see Materials and Methods). The procedure presented here on PMMA (both for 1 and 2 h of reaction) resulted in a density of primary amine groups that was twice as high as for the reaction described by Bulmus et al
) (Table ). The uniformity of primary amines on chemically treated slides was determined by immersion of a modified and non-modified PMMA surface into a 0.1 mg/ml FITC solution and by subsequently scanning the substrates. The fluorescence was homogeneous on activated PMMA sheets while the fluorescent signal was almost absent on non-modified PMMA surfaces (data not shown). The density of NH2
groups on the PMMA surface was four to five times lower than the values reported for silanization of glass or silica surfaces (32
) and 20-fold lower than the values reported by others for the modification of PMMA (6.85 nmol/cm2
). However, the values of primary amines reported here are still in excess when compared with the density of closely packed ssDNA strands (150 pmol/cm2
) oriented normal to the surface, considering a cross-sectional diameter of 20 Å for the ssDNA chain (20
Quantification of amino groups on chemically modified PMMA
The effect of immobilization kinetics, probe concentration, cross-linker types and chemical modifications procedures, on the density of DNA capture probes achieved on aminated PMMA sheets was investigated.
To find the conditions that resulted in the highest probe density on the surface, the kinetics of probe immobilization, for various probe concentrations, was investigated on aminated PMMA cross-linked with glutaraldehyde (Fig. C). The results showed that the attachment density reaches a maximum between 1 and 2 h of immobilization (Fig. A). The highest DNA capture probe density (12 ± 0.8 pmol/cm2) was obtained using a 20 µM DNA solution for spotting and 2 h of immobilizing time. One of the calibration curves derived from a dilution series of the aminated capture probes, on chemically aminated PMMA (according to Fig. A), is shown in Figure B. There is a strong linear correlation between spotted DNA concentration and obtained fluorescence in the standard curve.
Figure 2 Immobilization kinetics of oligonucleotides onto chemically modified PMMA substrate (according to the protocol in Fig. A) using different probe concentrations and immobilization times (A). NH2-DNA oligo-probes were immobilized at concentrations (more ...)
The chemical modification proposed by Bulmus et al
) and the one described here were evaluated by immobilization of aminated probes. The probes were attached for 2 h, on glutaraldehyde-activated surfaces with an initial spotted solution of 20 µM. Table shows that the presented protocol resulted in approximately the same density of immobilized probes as the protocol described by Bulmus et al
Surface density of immobilized aminated DNA probes and utilization of amine groups for immobilization on chemically modified PMMA and cross-linked with glutaraldehyde
To determine the contribution of the 5′-end reactive group of the DNA probes (NH2
or SH) to the immobilization density, PMMA substrates chemically modified by aminolysis were spotted with 20 µM solutions of DNA probes with or without NH2
or SH groups in the 5′ terminus (probe-Cy3, Table ). The resulting densities for non-functionalized DNA probes were very similar: 2.0 pmol/cm2
for the PMMA aminated by the protocol described by Bulmus et al
) and 1.9 pmol/cm2
for the PMMA modified with the chemistry presented here. The 5′-end covalently immobilized DNA was calculated by subtracting these values to the attachment densities obtained after immobilization of DNA probes with the reactive amino and thiol groups on the 5′-end terminus resulting in ~10 pmol/cm2
of specifically immobilized probes (Table ).
Sequence of oligonucleotides used
The effect of glutaraldehyde and sulfo-EMCS (Fig. D) cross-linkers on the immobilization density was evaluated. The immobilization density obtained was on average 2-fold higher for microarrays prepared with glutaraldehyde (homo-functional) as cross-linker, than with the sulfo-EMCS (hetero-functional) (Fig. ). The drawback of glutaraldehyde as cross-linker was that the substrates must be used within hours after cross-linking. In contrast, PMMA modified with the sulfo-EMCS has a longer shelf-life (data not shown).
Figure 3 Immobilized density of oligonucleotides on modified PMMA substrates using different cross-linkers and probe concentrations. The 5′-amino-labeled ssDNA probes (squares) or thiolated DNA probes (circles) were immobilized on PMMA surfaces cross-linked (more ...)
DNA surface coverage increases with the increasing DNA concentration of the probe solution, reaching a plateau at 10 µM of spotted DNA (Fig. ). As mentioned above, the theoretical coverage of a close-packed full monolayer of ssDNA is 150 pmol/cm2
, considering that the DNA molecules are cylindrical having a 20 Å diameter and oriented perpendicular towards to the plane of the surface. The coverage, presented in Figure , corresponds to ~7% of a DNA monolayer using amino-modified DNA probes and 3% for thiolated DNA probes, assuming that the maximum surface density of ssDNA is 150 pmol/cm2
. The chemically aminated PMMA presented here, contains 0.29 nmol/cm2
groups [or 0.16 nmol/cm2
for the PMMA modified as Bulmus et al
)], which assuming that the surface is flat results in spacing between adjacent amino groups of ~4.3 Å (or 8 Å). This distance is smaller than the size of the cross-linkers used in this work (10.5 Å, glutaraldehyde and 18 Å, sulfo-EMCS) or the diameter of the DNA helix (~18–20 Å). Hence, the cross-linker molecules cannot be attached to the surface at the full density of NH2
groups; consequently, the surface density of the immobilized molecules is determined by the dimension of the molecules and not the number of NH2
groups on the surface.
A major concern when preparing DNA microarrays is the accessibility and selectivity of the surface-bound probe for hybridization. The selectivity of duplex formation by the immobilized DNA on aminated PMMA slides, was demonstrated by comparing hybridization of a complementary and a non-complementary DNA strand to an immobilized NH2-DNA probe on the modified PMMA surface. Negligible fluorescence was obtained for hybridization with the non-complementary fluorescent DNA (Fig. A), demonstrating that unspecific hybridization did not occur. The signal-to-noise ratio was on average 90, calculated from the signal obtained from complementary hybridized DNA spots and the signal obtained from non-complementary hybridized spots. It should be mentioned that 0.2 µM DNA target (target-Cy5, Table ) saturated the hybridization signal, since 0.5 and 1 µM DNA target solutions did not increase the hybridization signal.
Figure 4 Specificity, kinetics and efficiency of hybridization on PMMA. The aminated PMMA was obtained by the chemistry presented in this report followed by cross-linking with glutaraldehyde. The 5′-aminated probes were immobilized for 2 h, washed and (more ...)
To understand the dynamics of microarray hybridization, the kinetics of a hybridization reaction on a PMMA array was followed over 24 h. The signal intensity from hybridization proceeds linearly during the first 2 h (Fig. B). After this initial stage, the hybridization signal reaches a steady state between 5 and 24 h. Furthermore, the hybridization signal increases with the amount of spotted DNA probes on the surface (Fig. B).
The rate of hybridization can be examined as a function of the immobilized DNA density by fitting the data to the equation:
is the initial hybridization rate, Vmax
is the maximum calculated hybridization rate, I
is the density of immobilized DNA probes and K
is the concentration of I
where the hybridization rate is half of the maximum (33
). By plotting the initial rate of hybridization (the linear part of the kinetic curves, Fig. B) for different amounts of immobilized capture probes it was possible to observe that the hybridization rate was dependent on the DNA concentration in the spot for low immobilization probe densities, but approaches a maximum above a density of 4–6 pmol/cm2
(Fig. C). This density is approximately similar to the 2 pmol/cm2
obtained previously on CMT-GAPS-coated glass slides (33
However, our result is based on a probe that is 18 bp long while the result on the CMT-GAPS slides was obtained using a 716 bp long probe. The longer probe on CMT-GAPS slides could explain why this slide has a slightly lower density at which the maximum hybridization rate is obtained, since, presumably, a larger probe takes up more space on the surface resulting in a crowding effect at lower densities than a small oligonucleotide.
The surface densities determined for covalently attached DNA and hybridized DNA were used to calculate the percent of covalently attached probes that participated in duplex formation (defined here as hybridization efficiency). There was an optimal immobilized surface DNA density for which the hybridization efficiency reached a maximum 4 pmol/cm2
(which corresponds to a 5 µM spotted solution) and above this density the efficiency decreased with the amount of probes present on the surface (Fig. D). This optimal immobilization density is in agreement with the density obtained for achieving the maximum rate of hybridization at the surface. By performing these two types of data analysis (rate and efficiency of hybridization), it can be concluded that above an attachment density of 4 pmol/cm2
, there is no gain in the percentage of hybrids formed on the PMMA surface. These results are also in agreement with previous studies (34
), that found that the maximum hybridization efficiency on a gold surface was obtained for a 3 pmol/cm2
density of immobilized DNA. This effect is probably due to repulsive electrostatic and steric interactions that increase with the probe density.
The hybridization efficiency, for the aminolysis modified PMMA (according to Fig. A), is almost identical for the two types of spacers (15 and 18%, for glutaraldehyde and sulfo-EMCS, respectively) using the same amount of immobilized DNA probes on the surface (2.5 pmol/cm2).
The hybridization signals obtained on chemically modified PMMA slides were compared, under the same experimental conditions, to the hybridization signals obtained on PMMA modified according to Bulmus et al
), silanized glass slides, commercial silylated glass and commercial plastic Euray™ slides (Fig. A). The hybridization signals obtained were similar for hybridization to silanized glass and chemically modified PMMA. The percentage of immobilized probes involved in hybridization was strongly dependent on the amount of capture probes present at the surface, for all the substrates studied (Fig. B). Above 2–4 pmol/cm2
the yield of hybridization did not increase even if the density of immobilized probes increased. High surface coverage of ssDNA probes did not consequently lead to the formation of more hybrids, since overloading the surface with probes may cause a crowding effect that can lower the accessibility of the surface-bound probes (34
). However, the maximum surface coverage for dsDNA on a surface has been determined to be 50 pmol/cm2
), which is 5-fold higher than the maximum number of probes immobilized on PMMA slides and on glass slides (10 pmol/cm2
). The crowding effect should thus be small, but still the efficiency of hybridized probes was <20%, for all the substrates analyzed, meaning that besides the density of probes, other parameters should be taken into account, like the thermodynamic equilibrium between the immobilized probes and the complementary target DNA in solution, and the spatial orientation of the probes on the surface, after the immobilization.
Figure 5 Comparison of hybridization signals obtained on different substrates and chemistries. The amino-labeled DNA probes were immobilized on glutaraldehyde-modified slides in the case of PMMA and silanized glass. On commercial slides (silylated glass and Euray™) (more ...)
The spot-to-spot variation was compared and evaluated with respect to spot size and fluorescence intensity after hybridization (Fig. C and D). On all the polymeric plastic substrates a ‘foot-print’ or halo was present around each spot (Fig. C), representing an outline of the pin printing tool, and probably due to differences in surface properties between the printing pin and the substrate surface. The spot size variation for the PMMA substrate modified by aminolysis was low [45 µm, coefficient of variance (CV) 2%], when compared with the variation observed for the other chemically treated substrates (180–225 µm, CV 5–17%), as shown in Figure D. Using a non-contact printing method (Packard Biochip Arrayer) uniform (135 µm, CV 5%) rounded spots were obtained (Fig. C), appearing without shrinking.
The fluorescence signal variation observed on aminolysed PMMA was 10%, while PMMA modified as described by Bulmus et al
), Euray™ slides and silanized glass substrates had an average variation between 20 and 30% (Fig. D). The PMMA background signal was similar to the glass substrates, but lower than the other modified PMMA and commercial plastic slides (data not shown).
The chemically aminated PMMA surfaces (protocol according to Fig. A) resulted in the highest hybridization efficiency after 5 h of hybridization for a probe DNA density of 4 pmol/cm2
. For these conditions the hybridized density and the yield of hybridization were 0.75 pmol/cm2
and 18%, respectively. Considering that 0.29 nmol/cm2
groups are present on the aminated surface after chemical modification, only 0.26% of these groups were used in hybridization. The efficiency of hybridization depends on (i) the number of methyl ester groups accessible in the PMMA, (ii) the efficiency of obtaining primary amino groups, (iii) the degree of derivatization with the cross-linker, (iv) the number of accessible aldehyde or maleimide groups to the modified DNA probes, (v) the attachment efficiency of oligonucleotides to the modified PMMA surfaces and (vi) the efficiency in the reaction of hybrid formation between immobilized ssDNA and target DNA. All these steps can lead to the low yields of hybridization observed. The values reported here for immobilization (9.75 pmol/cm2
) of DNA probes on chemically modified PMMA sheets (according to Fig. A), is 300-fold higher than the values previously reported on PMMA (33.1 fmol/cm2
). Apparently, the procedure described by Waddell et al
) resulted in high amounts of NH2
on the surfaces (6.85 nmol/cm2
); however, very few of these were involved in immobilization of DNA probes.