Effects of novel quasi-interpenetrating network/gold nanoparticles composite matrices on DNA sequencing performances by CE

Dan Zhou1

Yanmei Wang1

Runmiao Yang1

Wenlong Zhang1

Ronghua Shi2

1Department of Polymer Scienceand Engineering,University of Science andTechnology of China,Hefei, P. R. China

2School of Life Science,University of Science andTechnology of China,Hefei, P. R. China

Received February 2, 2007Revised April 19, 2007Accepted April 24, 2007

Research Article

Effects of novel quasi-interpenetratingnetwork/gold nanoparticles compositematrices on DNA sequencing performancesby CE

Gold nanoparticles (GNPs) with particle sizes of about 20, 40, and 60 nm were prepared andadded into a quasi-interpenetrating network (quasi-IPN) composed of linear polyacryl-amide (LPA) with different viscosity-average molecular masses of 1.5, 3.3, and 6.5 MDa andpoly-N,N-dimethylacrylamide (PDMA) to form polymer/metal composite matrices, respec-tively. These novel matrices could improve ssDNA sequencing performances due to inter-actions between GNPs and polymer chains and the formation of physical cross-linkingpoints as demonstrated by intrinsic viscosities and glass transition temperatures. Theeffects of the parameters in relation to quasi-IPN/GNPs matrices, such as GNP contents,GNP particle sizes, LPA molecular masses, and solution concentrations, on ssDNA se-quencing performances were studied. In the presence of GNPs, the separation had theadvantages of high resolution, speediness, excellent reproducibility, long shelf life and easyautomation. Therefore, less viscous matrix solutions (with moderate size GNPs) due tolower solution concentration and lower-molecular-mass LPA could be used to replace moreviscous solutions (without GNPs) due to higher solution concentration or higher-molecular-mass LPA to separate DNA, while the sieving performances were approximate even higher,which helped to achieve full automation especial for capillary array electrophoresis (CAE)and microchip electrophoresis (MCE).

Keywords:

CE / Composite sieving matrices / DNA sequencing / Gold nanoparticles / Quasi-interpenetrating network DOI 10.1002/elps.200700068

2998 Electrophoresis 2007, 28, 2998–3007

1 Introduction

CE, instead of classical slab-gel electrophoresis (SGE), isbecoming one of the most important techniques for theanalysis DNA and proteins [1–4]. Sieving matrices are veryimportant during the separation and sequencing of DNA byCE [5–7] and nongel sieving matrices (i.e., noncross-linkingpolymer solutions) [8–16] were widely employed [17, 18] inrecent years, which can be seen in the review [19]. Among

them, linear polyacrylamide (LPA) with high molecular massoffers great advantages in terms of sequencing ability andread length [11, 12]. However, high-molecular-mass LPA so-lution is very viscous and LPA has no self-coating ability toreduce the EOF and the adsorption of DNA on the capillarywall efficiently. On the contrary, poly-N,N-dimethyl-acrylamide (PDMA) has excellent self-coating ability butoffers poor sieving performance. No single existing homo-polymer solution can fully meet all expectations. The choiceof polymer has often been arbitrary and empirical [20], pri-marily because the mechanisms of DNA separation are notfully understood [3]; the details about mechanisms can beseen in the review [21]. Therefore, searching for low-viscositypolymer solutions with high sieving ability and self-coatingability still remains an important issue for high-throughputDNA analysis [22]. It usually takes a lot of time and effort,however, to test and develop a new polymer matrix [23].Recently, addition of certain additives [22–33] into low-vis-cous polymer solutions has proved to be a very efficient andsimple method to overcome the difficulty of filling capillariesand improve dsDNA separation performance, as described

Correspondence: Professor Yanmei Wang, Department of Poly-mer Science and Engineering, University of Science and Technol-ogy of China, Hefei 230026, P. R. ChinaE-mail: [email protected]: 186-551-3601592

Abbreviations: AM, acrylamide; DMA, N,N-dimethylacrylamide;GNPs, gold nanoparticle; LPA, linear polyacrylamide; PDMA,

poly(N,N-dimethylacrylamide); POP-6, performance optimizedpolymers; quasi-IPN, quasi-interpenetrating network; TEM,

transmission electron microscope; Tg, glass transition tempera-tures; TTE, Tris–TAPS–EDTA buffer

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2007, 28, 2998–3007 CE and CEC 2999

previously [34]. These examples indicate that the additivesthat promote chemical or physical cross-linking are requiredfor good resolutions [32]. However, the study on additives forssDNA sequencing is very deficient at present. So we aim atimprovement of the performances on ssDNA sequencing byusing additives.

Wang et al. [35] synthesized a noncross-linking quasi-interpenetrating network (quasi-IPN) formed by LPA withhigh molecular mass (up to 9.9 MDa) and PDMA as a high-performance ssDNA sequencing medium, which couldcombine the high sieving ability of LPA and the dynamiccoating ability of PDMA. In order to prepare LPA with highmolecular mass, a great quantity of expensive bis(2-ethyl-hexyl)sulfosuccinate (AOT) was used as an emulsifier. Inprevious paper [36], we prepared LPA with lower molecularmass (3.3 MDa) using a small amount of cheap sorbitanmonooleate (Span 80) emulsifier. To improve the ssDNA se-quencing properties using quasi-IPN formed by LPA withlower molecular mass and PDMA, we added about 40 nmgold nanoparticles (GNPs) into this quasi-IPN to form poly-mer/metal composite matrices. GNPs may help to stabilizethe sieving network, which is similar with the solutionsdescribed previously [37, 38]. The sequencing results onssDNA show that the performances of quasi-IPN/GNPs werehigher than those of quasi-IPN without GNPs.

Based on our previous work [36], in this study, in order tofurther investigate the effects of the composite matrices onssDNA sequencing performances and indicate the possiblefunctions of GNPs, we prepared quasi-IPN/GNPs compositematrices in various combinations of LPA (1.5, 3.3, and6.5 MDa) and GNPs (about 20, 40, and 60 nm) at differentGNPs contents and solution concentrations, and appliedthem to ssDNA sequencing, respectively. The effects of theparameters in relation to quasi-IPN/GNPs composite sievingmatrices, such as GNPs contents, GNPs particle sizes, LPAmolecular masses, and solution concentrations, on ssDNAsequencing performances were studied in detail by CE. Andthe possible functions of GNPs in DNA sequencing werediscussed. Because the content of PDMA in quasi-IPN wasvery low (about molar ratio of 30:1 for acrylamide/N,N-dimethylacrylamide (AM/DMA)) and PDMA was mainlyused for dynamic coating not for sieving, its content in quasi-IPN was not considered seriously. Additionally, the sequenc-ing performances of quasi-IPN/GNPs matrices were com-pared with those of commercial product performance opti-mized polymers (POP-6) and the reproducibility of sequenc-ing was investigated.

2 Materials and methods

2.1 Materials

The details of the reagents and BigDye Terminator kit V3.1sequencing standard DNA sample used in these experi-ments have been reported elsewhere [36]. In DNA sequenc-

ing systems, the 16TTE buffer (50 mM Tris/50 mM TAPS/2.0 mM EDTA in water) is recommended and traditionallyused to keep the pH value (,8.3) constant, and high con-centration of urea (such as 7 M) is employed in order toensure DNA denatured and keep DNA in single-strandedconformation.

2.2 Preparation and characterization of

quasi-IPN/GNPs

LPAs were synthesized by using inverse emulsion polymeri-zation of AM (20 g) in our laboratory [36, 39] according to theprotocols [10, 40] with some slight changes. Different amountsof initiators, i.e., ammonium persulfate (APS) and TEMED,were used for preparing LPA with different molecular masses:160 mL of 0.1 g/mL APS aqueous solution and 20 mL ofTEMED, 80 mL of 0.1 g/mL APS aqueous solution, and 10 mLof TEMED, as well as 40 mL of 0.1 g/mL APS aqueous solutionand 5 mL of TEMED for LPA with low (1.5 MDa), middle(3.3 MDa), and high (6.5 MDa) molecular mass, respectively.

In order to form quasi-IPN, three aliquots of 0.2 mL ofDMA were added into each of 50 mL of 1% w/v LPA aqueoussolutions with 1.5, 3.3, and 6.5 MDa [35, 36], respectively.The polymerization of DMA was initiated by APS andTEMED at 07C. The final products were signed as quasi-IPN1, quasi-IPN3, and quasi-IPN6 according to the molecu-lar mass of LPA, respectively.

Au colloid was prepared according to Frens [41] withslight modifications. Different particle sizes of GNPs wereprepared by the same procedure, the only change being inthe amount of trisodium citrate solution added. In three100 mL round-bound flasks equipped with condensers, eachof 50 mL of 0.01 wt.% HAuCl4 solution was added andheated to a boil with vigorous stirring. 0.85, 0.5, and 0.3 mLof 1 wt.% trisodium citrate solution were rapidly added intothese HAuCl4 solutions, and the prepared colloids weresigned as GNPs20 (,20 nm), GNPs40 (,40 nm), andGNPs60 (,60 nm) according to the particle sizes of GNPs,respectively.

To obtain composite matrices prepared in various com-binations of LPA with different molecular masses and GNPswith different particle sizes and contents, 0.25 and 2.0 mL ofabove prepared Au colloids (GNPs20, GNPs40, and GNPs60)were added into six aliquots each of 20 mL of 1% w/v quasi-IPN solutions (such as quasi-IPN1), respectively. The sixsolutions were precipitated in excess acetone and the final sixquasi-IPN/GNPs composites were filtered and dried undervacuum, signed as quasi-IPN1/GNPs20-1, quasi-IPN1/GNPs20-2, quasi-IPN1/GNPs40-1, quasi-IPN1/GNPs40-2,quasi-IPN1/GNPs60-1, and quasi-IPN1/GNPs60-2, respec-tively. The same did the quasi-IPN3 and quasi-IPN6.

The sieving matrix solutions were prepared by mixingthe quasi-IPN/GNPs with 16TTE buffer/7 M urea to thedesired concentrations (2.0–2.5% w/v). In order to compar-ison, quasi-IPN sieving matrix solutions without GNPs werealso prepared.


3000 D. Zhou et al. Electrophoresis 2007, 28, 2998–3007

LPAs, quasi-IPN, GNPs, and quasi-IPN/GNPs werecharacterized using an Ubbelohde viscometer (intrinsic vis-cosity [Z]), AVANCE 300 1H-NMR Spectrometer, Hitachi H-800 Transmission Electron Microscope (TEM), UV-2401PCUV–Vis Spectrophotometer, AAnalyst 800 Atomic Absorp-tion Spectrometer (AAS), and TA-50 Thermal Analyzer,respectively [36].

2.3 DNA sequencing by CE and data processing

Sequencing of standard DNA sample was performed on anABI 310 PRISM™ Genetic Analyzer (Perkin-Elmer, AppliedBiosystems Division, USA) with four-color LIF detection.The bare fused-silica capillary can be reused by filling thecapillary with new polymer solutions after treatment usingwater.

In order to quantify the separation performance of amatrix and compare it with other matrices, the resolutions(R) and two other parameters (separation selectivity S andseparation efficiency N) of selected nine pairs of DNA frag-ments with lengths of 105/106, 210/211, 250/251, 306/307,404/405, 524/525, 620/622, 744/746, and 938/942 baseswere calculated according to the equations [36, 42], respec-tively.

3 Results and discussion

3.1 Preparation and characterization of quasi-IPN/

GNPs

LPAs with different molecular masses were produced byusing different amounts of initiators through inverse emul-sion polymerization. The viscosity-average molecular massesof LPA were calculated from [Z] according to the Mark–Houwink equation [43, 44] to be 1.5, 3.3, and 6.5 MDa,respectively (Table 1).

Quasi-IPNs were formed by solution polymerization ofDMA in LPA aqueous solutions, which were noncross-link-ing networks different from traditional IPN cross-linkingnetworks [35]. The increase in the number of entanglementsand the presence of more extended polymer chains renderedthe quasi-IPN with a higher sieving ability, which has beendemonstrated by Chu’s group [35, 45, 46]. Typical 1H-NMRspectra of LPA and quasi-IPN in D2O can be seen in ourprevious paper [36]. The molar ratios of AM to DMA in threequasi-IPNs estimated from the integral peak area ratios ofthe peak at ,1.7 ppm (due to the methylene protons of LPAand PDMA) and peak at 3.0 – 3.1 ppm (due to the methylprotons of PDMA) were all about 30/1 (Table 1).

Table 1. Properties of LPA, quasi-IPN, and quasi-IPN/GNPs containing LPA with different molecular masses and GNPs with differentparticle sizes and contents

Intrinsicviscosity (mL/g)

D[Z]/[Z]a)

(%)Mr of LPA(MDa)

Molar ratioof AM/DMA

Glass transitiontemperature Tg (7C)

LPA1 397 – 1.5 – 192.80Quasi-IPN1 380 – 1.5 27 193.11Quasi-IPN1/GNPs20-1 404 6.32 1.5 27 193.26Quasi-IPN1/GNPs20-2 413 8.68 1.5 27 194.47Quasi-IPN1/GNPs40-1 391 2.89 1.5 27 193.62Quasi-IPN1/GNPs40-2 413 8.68 1.5 27 194.53Quasi-IPN1/GNPs60-1 390 2.63 1.5 27 193.27Quasi-IPN1/GNPs60-2 414 8.95 1.5 27 194.94LPA3 720 – 3.3 – 191.90Quasi-IPN3 717 – 3.3 30 192.98Quasi-IPN3/GNPs20-1 725 1.12 3.3 30 193.06Quasi-IPN3/GNPs20-2 772 7.67 3.3 30 194.14Quasi-IPN3/GNPs40-1 725 1.12 3.3 30 193.55Quasi-IPN3/GNPs40-2 767 6.97 3.3 30 194.70Quasi-IPN3/GNPs60-1 731 1.95 3.3 30 193.48Quasi-IPN3/GNPs60-2 770 7.39 3.3 30 194.36LPA6 1196 – 6.5 – 192.38Quasi-IPN6 1188 – 6.5 31 192.42Quasi-IPN6/GNPs20-1 1191 0.25 6.5 31 193.15Quasi-IPN6/GNPs20-2 1203 1.26 6.5 31 194.26Quasi-IPN6/GNPs40-1 1198 0.84 6.5 31 193.13Quasi-IPN6/GNPs40-2 1216 2.36 6.5 31 194.15Quasi-IPN6/GNPs60-1 1192 0.34 6.5 31 193.66Quasi-IPN6/GNPs60-2 1197 0.76 6.5 31 194.65

a) [Z] denotes intrinsic viscosities of quasi-IPN; D[Z] denotes the differences of intrinsic viscosities of quasi-IPN/GNPs and quasi-IPN;D[Z]/[Z] denotes increment ratios in intrinsic viscosities for quasi-IPN/GNPs relative to quasi-IPN.



During the preparation of Au colloids, citrate acted asboth a reducing agent and a capping agent [47] to avoidaggregation of GNPs, thus the surface of prepared GNPswas negatively charged with citrate. But after addition of avery small amount of GNPs into the polymer solution tomix uniformly and precipitation of the mixture using ace-tone, the negative charges of the surface of GNPs in com-posites were partly displaced or masked by neutral polymerchains, the charge density of GNPs maybe decreasedremarkably and GNPs were near neutral and stable inpolymer solution, similar to [28, 29]. Thus, GNPs with verylow negative charge had relatively weaker interaction withnegative DNA in TTE buffer at pH,8.3. The particle sizesof prepared GNPs could be controlled by the amount of tri-sodium citrate, and more trisodium citrate resulted in asmaller particle size of GNPs. So approximately sphericalisolated GNPs with mean diameters of about 20 nm(GNPs20), 40 nm (GNPs40), and 60 nm (GNPs60) areobserved according to the TEM images (Figs. 1A–C),respectively. It is found that GNPs in quasi-IPN polymersolution did not further agglomerate and dispersed evenly(Fig. 1D).

Figure 1. TEM images of (A) GNPs20 (,20 nm), (B) GNPs40(,40 nm), and (C) GNPs60 (,60 nm) in water, and (D) GNPs40(,40 nm) in quasi-IPN3 polymer solution.

Figure 2 shows UV–Vis spectra of the GNPs20, GNPs40,and GNPs60 in water. The curves reveal the characteristicmaximum absorbance wavelength for surface plasmon reso-nance (SPR) were 524, 529, and 532 nm, which were con-sistent with the presence of ,20, 40, and 60 nm GNPs,respectively.

The contents of the GNPs in quasi-IPN/GNPs compo-sites were measured by using AAS, therefore the contents ofthe GNPs in matrix solutions were estimated to be about1.25 mg/mL for quasi-IPN/GNPs-1 and 11.12 mg/mL forquasi-IPN/GNPs-2, respectively.

As can be seen from Table 1, the intrinsic viscosities ofquasi-IPNs and quasi-IPN/GNPs in dilution solutions wouldaugment a little with the contents of GNPs. The increase inintrinsic viscosity suggests the expansion of the polymer coiland the increase of apparent molecular mass because of theexistence of GNPs in polymer network. According to Dolníket al. [48], the potentially best sieving polymers are those withhigh intrinsic viscosity that can be used for a first selection ofsieving polymers before DNA sequencing. Moreover, theratios of increment in intrinsic viscosities for quasi-IPN/GNPs relative to quasi-IPN with 1.5 MDa LPA were largerthan those with 6.5 MDa LPA at the same GNPs content andparticle size as listed in Table 1. This means that the effects ofGNPs on short chain LPA were more significant than thoseon long chain one.

Thermal analysis (DSC) results (Table 1) show that theglass transition temperatures (Tg) increased slightly when asmall amount of GNPs were added into quasi-IPN systems,indicating that there were interactions between GNPs andthe polymer chains. Generally, the functional groups (i.e., –CN, –SH and –NH2) on polymer chains have high affinity forthe colloidal Au particles [49]. In quasi-IPN/GNPs, Au parti-cles were bound to the LPA chains through their interactionswith –NH2 of LPA and physical cross-linking points might beformed, so the free volume in polymer became smaller, thematrix networks became more robust, and [Z] and Tg

Figure 2. UV–Vis spectra of (A) GNPs20 (,20 nm), (B) GNPs40(,40 nm), and (C) GNPs60 (,60 nm) in water.



became higher. But it is noted that high molecular masses ofLPAs could hardly affect Tg when they were higher than acritical value, as can been seen from Table 1 that all of the Tg

of LPAs with different molecular masses were almost thesame.

3.2 DNA sequencing by CE and data analysis

Resolution comparison of DNA sequencing using 2.5% w/vquasi-IPN and quasi-IPN/GNPs with various GNP con-tents, GNP particle sizes, and LPA molecular masses at507C are shown in Fig. 3, where the resolutions of ninepairs of DNA fragments were calculated for the 21 matrices

and plotted against the base number. Obviously, with thesame LPA molecular mass, the sequencing abilities ofquasi-IPN/GNPs containing different GNPs contents andparticle sizes were all higher than those of quasi-IPN with-out GNPs.

3.3 Effects of GNP contents on sequencing

The separation of 744/746 DNA fragments could not beachieved using quasi-IPN1 (with LPA molecular mass being1.5 MDa), but addition of GNPs could effectively improveseparation performance, as shown in Fig. 3A. We can alsofound from Figs. 3B and C that when LPA molecular

Figure 3. Resolution versus base number in sequencing of Bigdye Terminator V 3.1 sequencing standard DNA sample by CE using (A)quasi-IPN1, quasi-IPN1/GNPs20-1, quasi-IPN1/GNPs20-2, quasi-IPN1/GNPs40-1, quasi-IPN1/GNPs40-2, quasi-IPN1/GNPs60-1, and quasi-IPN1/GNPs60-2; (B) quasi-IPN3, quasi-IPN3/GNPs20-1, quasi-IPN3/GNPs20-2, quasi-IPN3/GNPs40-1, quasi-IPN3/GNPs40-2, quasi-IPN3/GNPs60-1, and quasi-IPN3/GNPs60-2; (C) quasi-IPN6, quasi-IPN6/GNPs20-1, quasi-IPN6/GNPs20-2, quasi-IPN6/GNPs40-1, quasi-IPN6/GNPs40-2, quasi-IPN6/GNPs60-1, and quasi-IPN6/GNPs60-2; (D) quasi-IPN1, quasi-IPN1/GNPs40-1, quasi-IPN3, quasi-IPN3/GNPs40-1,quasi-IPN6, quasi-IPN6/GNPs40-1, and commercial POP-6. Sequencing conditions: solution concentration of prepared matrices, 2.5% w/v;effective/total length of bare fused-silica capillaries, 50/61 cm; id/od, 75/365 mm; sequencing electric field strength, 150 V/cm for our se-quencing matrices or 200 V/cm for POP-6; sequencing temperature, 507C; DNA electrokinetic injection, 41 V/cm for 30 s; anode buffer,16TTE; cathode buffer, 16TTE/7 M urea.



mass and GNP particle sizes were fixed, the resolutions ofquasi-IPN/GNPs were higher than those of quasi-IPNwithout GNPs, but the resolutions would decrease withfurther increase of GNPs content. In order to examine thereasons for better separation ability using quasi-IPN/GNPsmatrices, two parameters (selectivity S and separation effi-ciency N of nine pairs of DNA fragments) affecting the res-olution were calculated (data not shown). With the sameLPA molecular mass and GNP particle size, there was nosignificant change in selectivity with increasing the contentof GNPs [36]. At a solution concentration above C* (overlapconcentration), the linear polymer chains entangled oneanother to form a transient network with a certain meshsize (pore size). If the DNA fragment had a size larger thanthe mesh size, it would adjust itself to migrate through thenetwork like a snake, as described by the reptation model[50, 51]. On the other hand, the network would also makean “adjustment” to enlarge the mesh size by changing thepositions of the entanglements by a slide of the chains,resulting in a lower sieving ability [5]. However, it is clearlyshowed that the addition of GNPs into the quasi-IPN net-works could improve separation efficiency [36], which wasmainly originated from more stable networks due to theinteractions of GNPs with polymer chains, slower polymerchain disentanglement (longer pore lifetime) and moreextended chains of the networks [52]. Moreover, it is alsonoted that high efficiency was partially due to minimizedDNA adsorption on the capillary wall in the presence ofGNPs that were adsorbed on the wall [29]. As the content ofGNPs was increased, however, local aggregation of GNPs inpolymer network might occur. This incompatibility couldthen result in nonhomogeneous polymer–DNA interactionsand decrease the efficiency in sequencing [36], so the effi-ciency of quasi-IPN/GNPs-2 with higher GNP content waslower than that of quasi-IPN/GNPs-1. Depending on theselectivity and separation efficiency, the resolutions of quasi-IPN/GNPs-1and quasi-IPN/GNPs-2 were higher than thoseof quasi-IPN without GNPs. However, the resolutionswould decrease with increasing GNP content at the sameLPA molecular mass and GNP size in most case, as shownin Figs. 3A–C.

The presence of GNPs would also change migration timeof DNA fragments, for instance, quasi-IPN3, quasi-IPN3/GNPs40-1, and quasi-IPN3/GNPs40-2 at 507C resulted inabout 1000 bases being sequenced in 78, 73, and 71 min,respectively. On the one hand, the decrease in migrationtime in the presence of GNPs was likely because some GNPsadsorbed on the capillary wall surface and weakened theadsorption of DNA, leading to the decrease of EOF [53, 54]and thus the increase of DNA mobility. In addition, theinteractions between a very small amount of citrate and DNAmight shorten migration time [22]. On the other hand,migration time reduced mildly, indicating that only verysmall amount of GNPs adsorbed on the capillary inner walland thus the robust networks stabilized by GNPs wouldhardly be affected [36].

3.4 Effects of GNP sizes on sequencing

In order to study the effects of particle sizes of GNPs on DNAsequencing performances, GNPs with three particle sizes(about 20, 40, and 60 nm) were prepared by adjusting theamounts of trisodium citrate. It is observed from Figs. 3A–Cthat when LPA molecular mass and GNPs content werefixed, the effects of ,40 nm GNPs on resolutions were moresignificant than those of ,20 and ,60 nm GNPs no matterwhich quasi-IPN system was used. On the one hand, in thepresence of larger size of GNPs (,60 nm), LPA chains sur-rounding or adsorbing on the surfaces of GNPs are possiblynot long enough to protrude into solution [22], thus theinteraction between LPA chains reduced and networks wererelatively less stable, leading to loss of resolutions. Moreover,wider distribution and easier aggregation of larger GNPs(slightly turbid Au colloid could be observed in experiment)owing to smaller amount of citrate during preparation ofGNPs might also result in reduced resolutions. On the otherhand, too small GNPs (,20 nm) relative to long LPA chainscould not effectively act as cross-linking points, consequentlyresolutions decreased. Although the resolutions using ,20and ,60 nm GNPs were lower than those using ,40 nmGNPs, addition of GNPs ranging in diameter from 20 to60 nm into quasi-IPN matrices (with 1.5–6.5 MDa LPA)could improve DNA sequencing performances remarkably.

3.5 Effects of LPA molecular masses on sequencing

LPA is a very important component in these composite ma-trices and its molecular mass can determine the sievingperformances to a great extent. For further demonstration ofthe function of GNPs and better comparison of the sequenc-ing performances of quasi-IPN/GNPs (containing lower-molecular-mass LPA) with those of quasi-IPN (containinghigher-molecular-mass LPA), several quasi-IPN and quasi-IPN/GNPs systems with different LPA molecular mass (1.5,3.3, and 6.5 MDa) were prepared and used for DNA se-quencing analysis under the same sequencing conditions asin Fig. 3. From Fig. 3, it is found that quasi-IPN1 and quasi-IPN1/GNPs with lower-molecular-mass LPA (1.5 MDa) pos-sessed worse resolutions, and the DNA fragments of 938/942could not be separated by using these matrices even in thepresence of GNPs because lower molecular weight of LPA(1.5 MDa) formed less robust networks unfavorable to sepa-rate large DNA fragments. The resolutions would increasewith increasing the molecular mass of LPA, and the separa-tion of the DNA fragments of 938/942 could be achievedusing matrices with 3.3 or 6.5 MDa LPA.

As can be seen in Fig. 3A, addition of GNPs into quasi-IPN1 containing lower-molecular-mass LPA (1.5 MDa)could significantly improve sieving ability for both small andlarge DNA fragments. These results were mainly attributedto the following explanations. The mesh pore of polymernetworks would become smaller with addition of GNPs dueto the formation of physical cross-linking points, leading to



increased resolutions for small DNA fragments. At the sametime, more robust networks and higher apparent molecularmass due to addition of GNPs resulted in improved resolu-tions for large DNA fragments, for example, 744/746 DNApair could be separated after adding GNPs. As molecularmass of LPA increased, however, the effects of adding GNPsinto matrices on sequencing for large DNA fragmentsbecame weaker although the effects for small DNA frag-ments were still great (Figs. 3B and C). This was mainly be-cause a more robust network of quasi-IPN with greatersieving ability had been formed due to higher-molecular-mass LPA itself although in the absence of GNPs, in otherwords, addition GNPs could not show significant impactson the large DNA sequencing using quasi-IPN with higher-molecular-mass LPA, similar with the results of intrinsicviscosity from Table 1. But when molecular mass of LPA wastoo low (such as less than 0.6 MDa), even the presence ofGNPs could not promote the resolutions obviously (data notshown), which may be because it’s difficult for quasi-IPNitself with very-low-molecular-mass LPA to form efficientnetwork.

It is shown from Fig. 3D that the resolutions of quasi-IPN3/GNPs40-1 approximated those of quasi-IPN6 withoutGNPs, which further displays that in the composite sievingmatrices, GNPs interacted with polymer chains, and mightact as physical cross-linking points, thus increased apparentmolecular mass of quasi-IPN. Based on the separationmechanisms, these cross-linking points would help to formmore robust sieving matrix networks, and thus improvesieving properties. As shown in Table 1, the intrinsic viscos-ity of quasi-IPN3/GNPs40-1 was much lower than that ofquasi-IPN6. Although the polymer with higher intrinsic vis-cosity may be better sieving matrix [48], quasi-IPN3/GNPs40-1 with lower molecular mass and lower intrinsicviscosity can possess high sequencing performances close tothose of quasi-IPN6 due to the addition of GNPs into matri-ces. And the similar results can also be seen from Fig. 3Dthat the resolutions of quasi-IPN1/GNPs40-1 approximatedthose of quasi-IPN3 without GNPs. The use of quasi-IPN/GNPs can avoid the problems in relation to LPA with highmolecular weight such as difficult preparation and very highviscosity, and thus help for full automation. Moreover, lowerviscosity as a result of lower molecular mass resulted in lessmigration time of about 1000 base with close resolutions:72 min versus 78 min for using quasi-IPN1/GNPs40-1 versusquasi-IPN3, and 73 min versus 80 min for using quasi-IPN3/GNPs40-1 versus quasi-IPN6, respectively.

3.6 Effects of solution concentrations on sequencing

Figure 4 shows resolution comparison in DNA sequencingby CE using 2.0 and 2.3% w/v quasi-IPN3/GNPs40-1, and2.5% w/v quasi-IPN3 with the same molecular mass ofLPA at 507C, respectively. Quasi-IPN3/GNPs40-1 (2.0 and2.3% w/v) showed higher resolutions than 2.5% w/v quasi-IPN3, except for small DNA fragments (smaller than 306

Figure 4. Resolution versus base number in DNA sequencing byCE using 2.0 and 2.3% w/v quasi-IPN3/GNPs40-1, and 2.5% w/vquasi-IPN3 at 507C. Other sequencing conditions and DNA sam-ple as in Fig. 3.

bases) using 2.0% w/v quasi-IPN3/GNPs40-1 owing to lowersolution concentration and viscosity unfavorable to separatesmall DNA fragments, as shown in Fig. 4. Moreover, the for-mers resulted in about 1000 bases being sequenced muchfaster than the latter (about 61, 69, and 78 min, respectively).As a result of the interactions of GNPs with polymer chains,more dilute solutions with lower viscosity, such as 2.0 and2.3% w/v quasi-IPN3/GNPs40-1, possessed more excellentsieving performances in terms of resolution and migrationtime than 2.5% w/v quasi-IPN3 without GNPs. Hence, in thepresence of GNPs the less viscous solution due to lower so-lution concentration can be used to replace more viscous so-lution due to higher concentration without GNPs for auto-mation, while the sieving performances are approximateeven higher.

3.7 Comparison with commercially available POP-6

Finally, to demonstrate that quasi-IPN/GNPs are suitablematrices for DNA sequencing, their performances werecompared with those of the commercial product, POP-6from Applied Biosystems. For a proper comparison, se-quencing by using POP-6 was conducted under its individualconditions, which were suggested by the suppliers to opti-mize sequencing performances, for example, sequencingelectric field strength was 200 V/cm for POP-6 (while 150 V/cm for our sequencing matrices). Other sequencing condi-tions and DNA sample were the same as in Fig. 3. The reso-lutions by using POP-6 were better than those by usingquasi-IPN3, but worse than those by using quasi-IPN3/GNPs with the exception of small DNA fragments (smallerthan 250 bases) because higher solution concentration ofPOP-6 was helpful to separate small DNA fragments, asshown in Fig. 3D. And for the separation of larger DNAfragments, quasi-IPN3/GNPs showed much better resolu-



tions than POP-6, that is to say, quasi-IPN3/GNPs hadlonger read length. Moreover, the analysis time of quasi-IPN3/GNPs was also much shorter than that of POP-6, forexample, if the migration time of 620 bases was taken as aspeed criterion of DNA separation, the time was 107, 51, and47 min for POP-6, quasi-IPN3, and quasi-IPN3/GNPs40-1,respectively.

3.8 Reproducibility and shelf life

High reproducibility is important for DNA sequencing. Fig-ure 5 shows the plots of migration time and resolution versusbase number with SD error bars of migration time and reso-lution using quasi-IPN3/GNPs40-1 as sequencing matrix,respectively. When the same capillary system was used, theRSD of the migration time and resolution using quasi-IPN/GNPs containing LPA with molecular masses of 1.5, 3.3, and6.5 MDa and GNPs with particle sizes of about 20, 40, and60 nm at different contents as matrices for the first 15 runswere all less than 1.5 and 3.0%, respectively. The high repro-ducibility was attributed to the self-coating ability of PDMAand the adsorption of GNPs on the bare fused-silica capillaryinner wall, which could suppress EOF and avoid interactionsbetween DNA and capillary wall [29, 36]. In addition, the se-quencing resolutions of DNA sample did not show obviouschange after the quasi-IPN/GNPs matrix solutions werestored more than one year (data not shown), indicating thatthe composite matrix solutions were still very stable withtime and their shelf life was long.

Figure 5. Sequencing reproducibility of migration time and res-olution using 2.5% w/v quasi-IPN3/GNPs40-1 for the first 15 runsby CE at 507C. Sequencing matrix was replaced between runs.Other sequencing conditions and DNA sample as in Fig. 3.

4 Concluding remarks

GNPs (about 20, 40, and 60 nm) were prepared and addedinto quasi-IPNs composed of LPA (1.5, 3.3, and 6.5 MDa)and PDMA to form polymer/metal composite matrices for

DNA sequencing by CE. The effects of the parameters inrelation to quasi-IPN/GNPs sieving matrices, such as GNPcontents, GNP sizes, LPA molecular masses, and solutionconcentrations, on ssDNA sequencing performances werestudied in detail. The results show that with the same LPAthe resolutions of quasi-IPN/GNPs were higher than thoseof quasi-IPN without GNPs, but the resolutions would de-crease with further increasing GNP content, and theseparation would be faster after addition of GNPs. GNPsranging in diameter from about 20 to 60 nm, especiallyabout 40 nm GNPs, could all improve DNA sequencingperformances remarkably. Addition of GNPs into quasi-IPN1 containing 1.5 MDa LPA could significantly improvesieving ability for both small and large DNA fragments. Asmolecular mass of LPA increased, however, the effects ofGNPs on sequencing for large DNA fragments becameweaker. It is noted that the sieving ability of quasi-IPN/GNPs with lower-molecular-mass LPA and with GNPsapproximated those of quasi-IPN with higher-molecular-mass LPA and without GNPs. So the use of quasi-IPN/GNPs with lower-molecular-mass LPA could avoid theproblems in relation to LPA with higher-molecular-masssuch as difficult preparation and very high viscosity, andthus help for full automation. As a result of the interactionsof GNPs with polymer chains, more dilute solutions withlower viscosity possessed more excellent sieving perfor-mances in terms of resolution and migration time thanrelatively concentrated quasi-IPN without GNPs containingthe same LPA.

Higher sieving abilities of quasi-IPN/GNPs than those ofquasi-IPN could be explained as result of increase in separa-tion efficiency. In the presence of GNPs, the superior se-quencing efficiency was possible due to the interactions be-tween GNPs and polymer chains and the formation of phys-ical cross-linking points, as demonstrated by the studies ofintrinsic viscosity and DSC, which prevented the polymerchains from sliding away from each other and thus couldform relatively more stable “pore” sizes (more robust siev-ing matrix networks) and increase the apparent molecularmass of the matrices and thus their sieving properties.Moreover, high efficiency was partially due to minimizedDNA adsorption on the capillary wall in the presence ofGNPs that were adsorbed on the wall, leading to decrease ofEOF.

In conclusion, in the presence of the GNPs, the separa-tion had the advantages of high resolution, speediness, highefficiency, excellent reproducibility, long shelf life, and easyautomation. Thus, the less viscous matrix solutions (withmoderate-size GNPs) due to lower solution concentrationand lower-molecular-mass LPA could be used to replacemore viscous solutions (without GNPs) due to higher solu-tion concentration or higher-molecular-mass LPA, while thesieving performances were approximate even higher. Thesematrices seem able to combine optimal sieving ability withdynamic coating ability and with moderate viscosity. Thisshould make these matrices particularly interesting for full



automation of DNA separation in microfluidic systems, inwhich high-pressure loading of sieving matrices raises moredifficulties than in conventional capillaries [55].

Additionally, compared with POP-6, quasi-IPN/GNPswith molecular masses of LPA larger than 3.3 MDa showedbetter resolution (except for small DNA fragments), longerread length, and shorter migration time. Therefore, it is fur-ther demonstrated that this family of composite matricesseems very promising to be used for DNA sequencing inspite of the still incomplete study.

Optimization of other sequencing conditions, such aspH and ion strength of buffer, and electric field strength, willbe performed in the future to further improve the sequenc-ing performances and interpret the role of GNPs. Further-more, other new additives will be tried to promote ssDNAsequencing and their functions on sequencing performanceswill be studied.

We greatly acknowledge the support of this work by theNational Natural Science Foundation of China (grant no.50373040), the Foundation for Development of Talent of AnhuiProvince (grant no. 2005Z026), and the Scientific ResearchFoundation for the Returned Overseas Chinese Scholars, StateEducation Ministry.

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Documents

Effects of novel quasi-interpenetrating network/gold nanoparticles composite matrices on DNA sequencing performances by CE