novel quasi-interpenetrating network/gold nanoparticles composite matrices for dna sequencing by ce

Dan Zhou1

Yanmei Wang1

Wenlong Zhang1

Runmiao Yang1

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 August 4, 2006Revised November 13, 2006Accepted November 20, 2006

Research Article

Novel quasi-interpenetrating network/goldnanoparticles composite matrices for DNAsequencing by CE

In order to further improve ssDNA sequencing performances using quasi-interpenetratingnetwork (quasi-IPN) as a matrix composed of linear polyacrylamide (LPA) with lower vis-cosity-average molecular mass (3.3 MDa) and poly(N,N-dimethylacrylamide) (PDMA), goldnanoparticles (GNPs) were prepared and added into this quasi-IPN to form polymer/metalcomposite sieving matrices. The studies of intrinsic viscosity and differential scanningcalorimetry (DSC) on quasi-IPN and quasi-IPN/GNPs indicate that there were interactionsbetween GNPs and polymer chains. The sequencing performances on ssDNA using quasi-IPN and quasi-IPN/GNPs (with different GNPs concentrations) as sieving matrices werestudied and compared by CE at different temperatures. The results show that resolutions ofquasi-IPN/GNPs were higher than those of quasi-IPN without GNPs and approximatedthose of quasi-IPN composed of LPA with higher MW (6.5 MDa) and PDMA without GNPsin the bare fused-silica capillaries. Furthermore, the sequencing time of quasi-IPN/GNPswas shorter than that of quasi-IPN under the same sequencing conditions. The influencesof GNPs and sequencing temperature on the sequencing performances of ssDNA were alsodiscussed. The separation reproducibility of quasi-IPN/GNPs solution was excellent and itsshelf life was more than 8 months.

Keywords:

CE / Composite sieving matrices / DNA sequencing / Gold nanoparticles / Quasi-interpenetrating network DOI 10.1002/elps.200600488

1072 Electrophoresis 2007, 28, 1072–1080

1 Introduction

Separation and sequencing analysis of DNA is the key toreveal genetic codes. Fast, sensitive, automated, high-throughput and cost-effective separations, and sequencingtechniques are crucial for DNA analysis. Instead of classicalslab-gel electrophoresis (SGE), CE, including capillary arrayelectrophoresis (CAE) and microchip electrophoresis (MCE),is becoming one of the most important techniques for theanalysis of charged biomolecules such as DNA and proteins[1–3]. CE is superior to SGE because it offers the possibility

for full automation, higher sensitivity, less analysis time,lower consumption of chemicals and samples, and onlineoptical detection [4].

During the separation and sequencing analysis of DNAby CE, the sieving matrices are very important because theydetermine the migration behavior and the resolution of DNAduring separation [5–7]. In recent years, non-gel sieving ma-trices (i.e., noncross-linking polymer solutions) wereemployed in CE for their faster run time as well as relativelyfacile loading and replacement of the separation matrix be-tween runs, enabling an increased automation of DNA se-quencing [8, 9]. Common non-gel sieving matrices includelinear homopolymers (such as linear polyacrylamide (LPA)[10–14], poly(N,N-dimethylacrylamide) (PDMA) [15], poly-(ethyleneoxide) (PEO) [16], PVP [17], cellulose and its deriva-tives [18]), copolymers (random, graft, and block copolymers),mixtures, etc.; the details are described in our previous review[19]. Among them, LPA with high molecular weight offersgreat advantages in terms of sequencing ability and readlength [13, 14]. However, additional high-pressure injectingequipment or manual injection is generally needed to fill thecapillary and replace the solution because high molecularweight LPA solution is very viscous, which increases the an-alytic cost and makes it difficult to achieve an automatic

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; AOT, bis(2-ethylhexyl)sulfosuc-cinate; APS, ammonium persulfate; DMA, N,N-dimethylacryla-mide; DSC, differential scanning calorimetry; GNP, gold nanopar-ticle; LPA, linear polyacrylamide; PDMA, poly(N,N-dimethyl-acrylamide); quasi-IPN, quasi-interpenetrating network; TTE,

Tris-TAPS-EDTA buffer; UHP, ultrahigh-purity

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

Electrophoresis 2007, 28, 1072–1080 CE and CEC 1073

replacement of sieving matrices. Another shortcoming ofLPA is that the capillary wall needs pretreatment before usedue to no self-coating ability to reduce efficiently the EOF andthe adsorption of DNA on the capillary wall. On the otherhand, PDMA has the best self-coating ability among thecommon matrices but offers poorer sieving performance dueto its higher hydrophobicity. Although a lot of polymer solu-tions have been developed and tested as sieving matrices forCE, no single existing homopolymer solution can fully meetall the expectations. The choice of polymer has often beenarbitrary and empirical [20], primarily because the mechan-isms of DNA separation are not fully understood [2], and thedetails about mechanisms can be seen in the review [21].Therefore, searching for low-viscosity polymer solutions withhigh sieving ability and self-coating abilities still remains animportant issue for high-throughput DNA analysis [22].

However, testing and developing a new polymer matrixusually takes a lot of time and effort because the processinvolves polymer molecular architecture and topology, elec-tric field, and solution conditions [23]. Recently, addition ofcertain additives (such as polyhydroxy compounds [24, 25],montmorillonite clay [23], gold nanoparticles (GNPs) [22,26–30]) into low-viscosity polymer solutions has been provedto be a very efficient and simple method to overcome thedifficulty of filling capillaries and improve the DNA separa-tion performance, as described previously [31]. Huang et al.[22, 26, 27] used poly(ethylene oxide) (PEO) solutions con-taining GNPs as sieving matrices for dsDNA fragmentseparation, and achieved reproducible, rapid and high-reso-lution dsDNA separations. But the study of additives forssDNA sequencing is very deficient at present.

Recently, Wang et al. [32] designed, synthesized, andtested a noncross-linking quasi-interpenetrating network(quasi-IPN) formed by LPA with high molecular mass (up to9.9 MDa) and PDMA as a novel high-performance ssDNAsequencing medium by CE, which can combine the high-sieving ability of LPA and the dynamic coating ability ofPDMA. In order to prepare LPA with high molecular weight,the inverse microemulsion polymerization was utilized anda great quantity of bis(2-ethylhexyl)sulfosuccinate (AOT) wasused as emulsifier. However, AOT was expensive and its pu-rification process was tedious. Furthermore, it was difficultto remove all of AOT from the final product, which had anegative effect on ssDNA sequencing.

In this work, we prepared LPA with lower molecularmass (3.3 MDa) using a small amount of cheap sorbitanmono-oleate (Span 80) as an emulsifier inspired by Goetzin-ger et al. [12] through a simple reverse emulsion polymeri-zation. In order to further improve the ssDNA sieving prop-erties using quasi-IPN formed by LPA with lower molecularweight and PDMA, we tried to add GNPs, which are inertnoble metals capable of existing in the unoxidized state at thenanoscale and offering a unique surface chemistry and weresynthesized through the reaction of HAuCl4 with trisodiumcitrate, into this quasi-IPN to form polymer/metal compositematrices. The affinity between GNPs and polymer chains

was studied through the measurements of intrinsic viscosityand thermal property of quasi-IPN and quasi-IPN/GNPs.GNPs may help to stabilize the sieving network, which issimilar to solutions described previously [33, 34]. The se-quencing performances on ssDNA using quasi-IPN andquasi-IPN/GNPs (with different GNP concentrations) assieving matrices were studied and compared by CE at differ-ent temperatures. The influences of GNPs and sequencingtemperature on the separation performances of DNA werediscussed. The reproducibility and shelf life of quasi-IPN/GNPs were also investigated.

2 Materials and methods

2.1 Materials

All water used in this experiment was deionized water anddistilled three times prior to use. N,N-Dimethylacrylamide(DMA) was purchased from Aldrich Chemical Company(Milwaukee, WI, USA) and distilled under reduced pres-sure before use. Acrylamide (AM), ammonium per-sulfate (APS), TEMED, HAuCl4?4H2O, trisodium citratedihydrate, and other reagents were obtained from Sino-pharm Chemical Reagents (Shanghai, China). AM wasrecrystallized twice from chloroform. APS and trisodiumcitrate dihydrate were recrystallized from water, respectively.Span 80 (Fluka Chemie, Buchs, Switzerland) was used as anemulsifier. Kerosene was washed orderly using concentratedH2SO4, H2O, NaOH aqueous solution and H2O, dried usingCaCl2, and then distilled. TAPS, Tris, urea, and EDTA werepurchased from Sigma–Aldrich (St. Louis, MO, USA); theywere of electrophoresis or analytical grade and were usedwithout further purification. Electrophoresis buffer was16TTE (Tris-TAPS-EDTA buffer) (50 mM Tris/50 mMTAPS/2.0 mM EDTA in water) for the anode and 16TTE/7 M urea for the cathode, which were prepared by dissolvingthe salts in water to known concentrations. All the solutionswere filtered by Millipore membrane filter (0.45 mm poresize) before use.

BigDye Terminator kit V3.1 sequencing standard DNAsample and formamide (Hi-Di™) were purchased fromApplied Biosystems (Foster City, CA, USA). Prior to se-quencing, 100 mL formamide was added to a tube containingthe lyophilized sequencing standard DNA. The sample wasmixed thoroughly on a vortex mixer and centrifuged at3000 rpm for 60 s, then denatured by heating for 2 min at957C and quickly chilled on ice.

2.2 Preparation of quasi-IPN/GNPs

LPA was synthesized by using inverse emulsion polymeri-zation in our laboratory [35], according to protocols [12, 36]with some slight changes. Span 80 (2.47 g), AM (20 g), andH2O (30 g) were added into a 250-mL four-neck, round-bot-


1074 D. Zhou et al. Electrophoresis 2007, 28, 1072–1080

tom flask equipped with a mechanical stirrer, a reflux con-denser, a nitrogen inlet/outlet tube, and a feeding funnel.Purified kerosene (50 mL) was added dropwise into the mix-ture. In order to remove oxygen, the reaction system waspurged continually with ultrahigh-purity (UHP) nitrogen(with a purity of 99.99%) for 1 h under a stirrer speed of500 rpm. Forty microliters of 0.1 g/mL APS aqueous solu-tion and 4.5 mL TEMED were used as initiator. The reactionwas performed at 227C for 24 h with constant stirring undercontinuous flow of UHP nitrogen. After polymerization,pure LPA was obtained by the precipitation of the emulsionin excess acetone under stirring and washing the precipitateseveral times with acetone. The final product was filtered anddried under vacuum.

In order to form quasi-IPN, 0.2 mL of DMA was addedinto 50 mL of 1% w/v LPA aqueous solution [32]. The reac-tion system was then purged by UHP nitrogen for 1 h. Thepolymerization of DMA was initiated by adding 50 mL of0.1 g/mL APS aqueous solution and 8 mL of TEMED at 07C,and allowed to proceed to completion for 24 h with constantstirring of 50 rpm under continuous flow of UHP nitrogen.The final product was precipitated in an excess of acetoneand dried under vacuum.

Before preparation of Au colloid, all the glassware wasthoroughly cleaned in aqua regia, then rinsed using water,and dried. Au colloid was prepared according to Frens et al.[22, 37] with slight modifications. In a 100-mL round-boundflask equipped with a condenser, 50 mL of 0.01 wt.% HAuCl4

was heated to a boil with vigorous stirring. One weight-per-cent of 0.5 mL of trisodium citrate was rapidly added intothis solution, and the solution changed color from pale-yel-low to blue and then to red-violet, indicating the formation ofthe GNPs. Boiling continued for an additional 10 min, theheating source was removed, and the colloid was stirred foranother 15 min and cooled to room temperature.

To prepare composite matrices containing GNPs withdifferent concentrations, 0.25 and 2.0 mL of above-preparedAu colloids were added into two aliquots each of 20 mL of 1%w/v quasi-IPN solutions, respectively. After the formation ofcompletely homogeneous mixtures, the two solutions wereprecipitated in excess acetone under stirring and the pre-cipitate was washed several times with acetone. The final twoquasi-IPN/GNPs composites were filtered and dried undervacuum, signed as quasi-IPN/GNPs-1 and quasi-IPN/GNPs-2, respectively.

The sieving matrix solutions were prepared by mixingthe quasi-IPN/GNPs with 16TTE buffer/7 M urea to thedesired concentration (2.5% w/v). In order to compare,quasi-IPN sieving matrix solution without GNPs was alsoprepared.

2.3 Characterization

Intrinsic viscosities [Z] of LPA, quasi-IPN, and quasi-IPN/GNPs were measured in 0.1 mol/L NaCl aqueous solutionsat 307C using an Ubbelohde viscometer. The viscosity-aver-

age molecular weight (Mn) of LPA was calculated from thefollowing Mark–Houwink equation [38, 39]:

[Z] = 9.3361023Mn0.75 (mL/g) (1)

1H-NMR spectra of LPA and quasi-IPN with D2O as the sol-vent were recorded by using an AVANCE 300 spectrometer(BRUKER BIOSPIN AG, Switzerland), respectively. The AMto DMA ratio in quasi-IPN was estimated from the integralpeak area ratio of the selected peaks.

The size and shape of GNPs were determined by a HitachiH-800 Transmission Electron Microscope (Hitachi High-Tech-nologies Corp., Tokyo, Japan). The sample for TEM observa-tions was prepared by dropping the sample solution onto cop-per grids. After deposition, any remaining solution was wickedaway, and the grids were dried in room temperature.

A UV-2401PC UV-Vis spectrophotometer (ShimadzuCorp., Japan) was used to measure the surface plasmonresonance (SPR) band of GNPs and quasi-IPN/GNPs inwater. An Analyst 800 Atom Absorption spectrophotometer(AAS) (Perkin-Elmer, USA) was used to measure the con-tents of GNPs in the two quasi-IPN/GNPs composites.

The differential scanning calorimetry (DSC) thermo-grams of quasi-IPN and quasi-IPN/GNPs were recordedusing TA-50 thermal analyzer (Shimadzu). The sampleswere heated from room temperature to 2307C at a heatingrate of 107C/min and nitrogen was used as atmosphere.

2.4 DNA sequencing by CE

Sequencing of Bigdye Terminator V3.1 standard DNA sam-ple was performed on an ABI 310 PRISM™ Genetic Analyzer(Perkin-Elmer, Applied Biosystems Division) with four-colorLIF detection. LIF detection was based on excitation by the488 nm line of Ar-ion laser with 10 mW power and the fluo-rescence emission were detected at about 540, 570, 595, and625 nm. The bare fused-silica capillaries (Polymicro Tech-nologies, Phoenix, AZ, USA) with id/od of 75/365 mm andeffective/total length of 50/61 cm were used without anycoating in our experiments. A detection window of 6 mmwidth was opened at 11 cm from the anode end by strippingoff the polyimide coating with a razor blade. Other sequenc-ing conditions: DNA sequencing electric field strength,150 V/cm; DNA electrokinetic injection into the capillary,41 V/cm for 30 s; anode buffer, 16TTE; cathode buffer,16TTE/7 M urea; sequencing temperature, 50 or 607C; so-lution concentration, 2.5% w/v. The capillary can be reusedby filling the used capillary with new polymer solutions aftertreatment using water.

2.5 Data processing

Since base-calling software of ABI 310 was not suitable forour sequencing matrices, raw LIF data were transformedthrough the ABI-Browser software and Origin 7.5 software



(Microcal, Northampton, MA, USA) was used to extract sin-gle color data (e.g. Green-track, base A) from transformeddata, which were fitted into Gaussian peaks by using Peak-Fit™ 4.06 software (SPSS, Chicago, USA). All the peak fit-tings had r2 .0.99. In order to quantify the separation per-formance of a matrix and compare it with other matrices, theresolutions (R) of selected nine pairs of DNA fragments withlengths of 105/106, 210/211, 250/251, 306/307, 404/405,524/525, 620/622, 744/746, and 938/942 bases were calcu-lated according to the following equation [40]:

R = 2 (t2 2 t1)/[1.6996(w2 1 w1)] (2)

where t and w are migration time and full peak width at halfheight (FWHM) of selected peaks, respectively, which can beobtained from sequencing electropherogram using PeakFit.There are two parameters (separation selectivity S andseparation efficiency N) affecting resolution. R, S, and Nhave the following relationship:

R = (SN1/2)/4 (3)

The selectivity and separation efficiency can be calculated bythe following two equations:

S = 2(t2 2 t1)/(t2 1 t1) (4)

N = 5.54 (t/w)2 (5)

As R and S are directly related to the size difference of twofragments (N1 2 N2), where N1 and N2 are base numbers ofDNA fragments, R/(N1 2 N2) and S/(N1 2 N2) are used herefor a proper comparison of different fragment pairs, i.e., Rand S of 620/622, 744/746, and 938/942 pairs are divided by2, 2, and 4, respectively.

3 Results and discussion

3.1 Preparation and characterization of

quasi-IPN/GNPs

LPA with lower molecular weight was produced usinginverse emulsion polymerization in our laboratory. Advan-tages of inverse emulsion polymerization over other poly-merizations include isolation of the domains of chemicalreaction, better temperature control, relatively low poly-dispersity of the polymer product, and low-viscosity reactionproducts containing a high-mass fraction of polymer [36]. Byadding the low-viscosity emulsion to acetone, LPA was pre-cipitated, while all other reaction components, includingresidual monomer, remained in solvents. The viscosity-aver-age molecular mass of LPA was calculated from [Z] accord-ing to Eq. (1) to be 3.3 MDa. 1H-NMR spectra of LPA in D2Ocan be seen in Fig. 1.

Quasi-IPN was formed by solution polymerization ofDMA in LPA aqueous solution, which was a noncross-link-ing network different from the traditional IPN cross-linkingnetworks [32]. On the one hand, the formation of quasi-IPNdramatically increased the entanglements between LPA andPDMA, resulting in a stabilized network. On the other hand,the incompatibility of LPA and PDMA would force them tobe separated from each other, resulting in more extendedpolymer chains. The increase in the number of entangle-ments and the presence of more extended polymer chainsrendered the quasi-IPN with a higher sieving ability, whichhas been demonstrated by Chu and co-workers [32, 41, 42].Figure 1 also shows typical 1H-NMR spectra of quasi-IPN inD2O. The methylene protons of LPA and PDMA combinedtogether had a chemical shift of ,1.7 ppm (peak a). Themethylidyne protons of LPA and PDMA had chemical shiftsof ,2.3 (peak b) and ,2.7 ppm (peak c), respectively. Themethyl protons of PDMA had chemical shifts from 3.0 to3.1 ppm (peak d) and were split into three peaks. The AM toDMA molar ratio in quasi-IPN estimated from the integralpeak area ratio of peak a and d was about 30/1.

During the preparation of Au colloids, citrate acts both asa reducing agent and as a capping agent [43] to avoid aggre-gation of GNPs. Approximately spherical isolated GNPs witha mean diameter of 40 nm are observed, according to theTEM images (the images are not shown).

The position of the SPR band is sensitive to particle sizeand shape as well as to the optical and electronic propertiesof the particle surroundings. Figure 2 shows UV-Vis spectraof the citrate-capped GNPs (Au colloids) and quasi-IPN/GNPs-2. Both curves reveal that the characteristic maximumabsorbance wavelength for SPR was 529 nm, which wasconsistent with the presence of the 40 nm GNPs. From Fig-ure 2, it is found that GNPs in quasi-IPN did not furtheragglomerate because there was no obvious difference inmaximum absorbance wavelength between Au colloids andquasi-IPN/GNPs.

Figure 1. 1H-NMR spectra of LPA and quasi-IPN in D2O.



Figure 2. UV-Vis spectra of GNPs and quasi-IPN/GNPs-2.

The contents of the GNPs in quasi-IPN/GNPs composites(measured by using AAS) and the concentrations of the GNPsin matrix solutions (calculated) are shown in Table 1. Theintrinsic viscosities of quasi-IPN and quasi-IPN/GNPs indilute solutions increased slightly with the concentrations ofGNPs (Table 1). The increase in intrinsic viscosity suggeststhe expansion of the polymer coil and the increase in apparentmolecular mass because of the existence of GNPs in polymernetwork. Dolník et al. [44] once studied the relationship be-tween intrinsic viscosity and sieving performance, andbelieved that the potentially best sieving polymers are thosewith high intrinsic viscosity which can be used for a firstselection of sieving polymers before DNA sequencing.

In order to further demonstrate the interactions of GNPswith polymer chains, thermal analysis was performed. Fig-ure 3 is the DSC thermogram of quasi-IPN, quasi-IPN/GNPs-1, and quasi-IPN/GNPs-2 and shows that the glasstransition temperature (Tg) increased slightly when a smallamount of GNPs were added into quasi-IPN system, indi-

Table 1. Properties of quasi-IPN, quasi-IPN/GNPs-1, quasi-IPN/GNPs-2, and quasi-IPN-H

Intrinsicviscosity[Z] (mL/g)

GNPcontents incomposites(mg/g)

GNP con-centrationsin solutions(mg/mL)

Quasi-IPNa) 717 – –Quasi-IPN/GNPs-1 725 50 1.25Quasi-IPN/GNPs-2 767 445 11.12Quasi-IPN-Hb) 1188 – –

a) Viscosity-average molecular mass of LPA, 3.3 MDa; mole ratioof AM/DMA, 30/1.

b) Viscosity-average molecular mass of LPA, 6.5 MDa (the prep-aration method was the same as quasi-IPN, but only the ratioof the monomer to initiator was changed); mole ratio of AM/DMA, 30/1.

Figure 3. DSC thermograms of quasi-IPN, quasi-IPN/GNPs-1, andquasi-IPN/GNPs-2.

cating that there were interactions between GNPs and thepolymer chains. Generally, the functional groups (i.e., 2CN,2SH and 2NH2) on polymer chains have high affinity forthe colloidal Au particles [45]. In quasi-IPN/GNPs, Au parti-cles were bound to the LPA chains through its interactionswith 2NH2 of LPA and physical cross-linking points mightbe formed. So, the matrix networks became more robust, and[Z] and Tg became higher.

3.2 DNA sequencing by CE and data analysis

Figure 4 shows the partial four-color (base C, T, A, G) elec-tropherograms of DNA sample using quasi-IPN/GNPs-1 assequencing matrix at 507C. Resolution comparison of singlecolor (e.g. Green-track, base A) of DNA sequencing usingquasi-IPN, quasi-IPN/GNPs-1, and quasi-IPN/GNPs-2 at507C and quasi-IPN/GNPs-2 at 607C is shown in Fig. 5,where the resolutions of nine pairs of DNA fragments werecalculated for the three matrices at different temperature andplotted against the base number. Obviously, the sequencingability of quasi-IPN/GNPs was better than that of quasi-IPNwithout GNPs. Quasi-IPN, quasi-IPN/GNPs-1, and quasi-IPN/GNPs-2 at 507C, and quasi-IPN/GNPs-2 at 607C resultedin 1000 bases being sequenced in about 78, 73, 71, and65 min, respectively (Fig. 6).

3.3 Influence of GNPs

In order to further examine the reasons for better separationability using quasi-IPN/GNPs matrices, two parameters(selectivity and separation efficiency of nine pairs of DNAfragments) affecting the resolution were calculated and plot-ted against the base number (figures are not shown).

Selectivity is defined as the mobility difference betweentwo peaks divided by both the average mobility and the DNAsize difference, which has been used to describe how far two



Figure 4. Partial four-color (baseC, T, A, G) electropherograms ofBigdye Terminator V 3.1 se-quencing standard DNA sampleusing 2.5% w/v quasi-IPN/GNPs-1 by CE at 507C. Sequencingconditions: effective/total lengthof bare fused-silica capillaries,50/61 cm; id/od, 75/365 mm; se-quencing electric field strength,150 V/cm; DNA electrokineticinjection, 41 V/cm for 30 s;anode buffer, 16TTE; cathodebuffer, 16TTE/7 M urea.

Figure 5. Resolution vs. base number in DNA sequencing by CEusing 2.5% w/v quasi-IPN, quasi-IPN/GNPs-1, quasi-IPN/GNPs-2,and quasi-IPN-H at 507C, and quasi-IPN/GNPs-2 at 607C. Othersequencing conditions and DNA sample as in Fig. 4.

fragments can be separated from each other [3]. It is observedthat all the matrices had higher selectivity less than about300 bases, after which the selectivity began to decrease. Avery slight decrease for low and high base numbers could bedetected when a small amount of GNPs was added intoquasi-IPN, and there was no significant change in selectivitywith an increase in the concentration of GNPs.

The separation efficiency (theory plate numbers permeter) implies the peak-broadening effect since the peakwidth is involved. At a solution concentration above C*(overlap concentration), the linear polymer chains entangledone another to form a transient network with a certain mesh

size (pore size). If the DNA fragment has a larger size thanthe mesh size, it will adjust itself to migrate through it like asnake, as described by the reptation model [46, 47]. On theother hand, the network would also make an “adjustment” toenlarge the mesh size by changing the positions of theentanglements by a slide of the chains, resulting in a lowersieving ability [5]. However, it is clearly shown that the addi-tion of GNPs into the quasi-IPN networks could improve theseparation efficiency, which was mainly originated frommore stable networks due to the interactions of GNPs withpolymer chains, slower polymer chain disentanglement(longer pore lifetime) and more extended chains of the net-works [48]. Moreover, it is also noted that high efficiency waspartially due to minimized DNA adsorption on the capillarywall in the presence of GNPs that were adsorbed on the wall[29]. However, as the concentration of GNPs was increased,local aggregation of GNPs in polymer network might occur.This incompatibility could then result in nonhomogeneouspolymer–DNA interactions and decrease the efficiency of se-quencing of larger DNA fragments, so the efficiency of quasi-IPN/GNPs-2 with higher GNP concentration was lower thanthat of quasi-IPN/GNPs-1 for larger DNA fragments.

Depending on the selectivity and separation efficiency,the resolutions of quasi-IPN/GNPs-1 and quasi-IPN/GNPs-2are higher than that of quasi-IPN without GNPs. However,the resolutions would decrease with increasing GNPs con-centration for larger DNA fragments, as shown in Fig. 5.

For better comparison and further demonstration of thefunction of GNPs, quasi-IPN composed of LPA with highermolecular weight (6.5 MDa) and PDMA (signed as quasi-IPN-H) was prepared, characterized (Table 1), and used forDNA sequencing under the same sequencing conditions as



Figure 6. Last parts of electro-pherogram (Green-track, baseA) in DNA sequencing by CEusing 2.5% w/v (a) quasi-IPN, (b)quasi-IPN/GNPs-1 and (c) quasi-IPN/GNPs-2 at 507C, and (d)quasi-IPN/GNPs-2 at 607C. Othersequencing conditions and DNAsample as in Fig. 4.

in Fig. 4. The results show that the resolutions of quasi-IPN/GNPs-1 are close to those of quasi-IPN-H with LPA(6.5 MDa) and without GNPs (Fig. 5), which further displaythat in the composite sieving matrices, GNPs interact withpolymer chains and might act as physical cross-linkingpoints, thus increasing the apparent molecular weight ofquasi-IPN. Based on separation mechanisms, these cross-linking points would help to form more robust sievingmatrix networks, and thus improve sieving properties. It isshown from Table 1 that the intrinsic viscosity of quasi-IPN/GNPs-1 (725 mL/g) is much lower than that of quasi-IPN-H(1188 mL/g). Although the polymer with higher intrinsicviscosity may be better sieving matrix [44], quasi-IPN/GNPs-1 with lower molecular weight and lower intrinsic viscositycan possess high sequencing performances close to those ofquasi-IPN-H due to the addition of GNPs into matrix. Theuse of quasi-IPN/GNPs can avoid the problems of LPA withhigh molecular weight such as preparation and very highviscosity, and thus help for full automation. Moreover, themigration time of quasi-IPN/GNPs-1 was shorter than thatof quasi-IPN-H (73 min vs. 80 min).

Quasi-IPN, quasi-IPN/GNPs-1, and quasi-IPN/GNPs-2 at507C resulted in about 1000 bases being sequenced in 78, 73,and 71 min, respectively (Fig. 6). On one hand, the decreasein migration time in the presence of GNPs was likely becausesome GNPs adsorbed on the capillary wall surface and wea-kened the adsorption of DNA, leading to the decrease in EOF[49, 50] and thus the increase in DNA mobility. In addition,the interactions between citrate and DNA might shorten themigration time [22]. On the other hand, migration timereduced mildly, indicating that only very small amount ofGNPs adsorbed on the capillary inner wall and thus therobust networks stabilized by GNPs would hardly be affected.

3.4 Influence of temperature

It is observed that there was no significant change in selec-tivity using quasi-IPN/GNPs-2 as matrices when the tem-perature was increased, although a slight decrease for lowbase numbers and slight increase for high base numberscould be detected. However, the separation efficiencydecreased significantly with an increase in temperature from50 to 607C. Higher thermal diffusion and shorter relaxationtime (blob lifetime) of the network at higher temperaturemight explain this phenomenon [51]. Moreover, hydrophobicDNA–polymer interactions due to the increase in the tem-perature could lead to band broadening and lower efficiency.According to the changes of selectivity and separation effi-ciency, the overall resolution decreased with increasing tem-perature, as shown in Fig. 5.

It has been shown by Dovichi and co-workers [52] andKarger and co-workers [53] that elevated temperatureincreased the read length in DNA sequencing by CE usingLPA as a separation medium [51]. In the same way, it isobserved that the sequencing size limit increased with highertemperatures using quasi-IPN/GNPs-2 as matrices (Figs. 6cand d). Elevated temperature should induce thermal energyinto the DNA molecules, which would reduce the extent offield alignment by DNA molecules and then shift onset ofbiased reptation DNA fragments toward higher base numbers[54, 55]. Higher temperatures also resulted in faster separationdue to decrease in solution viscosity, as shown in Figs. 6c and d.

3.5 Reproducibility

High reproducibility is important for DNA separation. Gen-erally, with an increase in run time, EOF will slowly show up,



making the migration time longer and the resolution poorer[20]. Migration time versus base number with SD error bars ofmigration time using quasi-IPN/GNPs-1 as sequencingmatrix was plotted (figure is not shown). When the samecapillary system was used, the RSD of the migration time forthe first 15 runs was less than 1.5% and there was no signif-icant loss of resolution. The reproducibility of matrix washigh, because the self-coating ability of PDMA and theadsorption of GNPs on the bare fused-silica capillary innerwall could suppress EOF and avoid interactions betweenDNA and capillary wall [29].

In order to evaluate the shelf life of quasi-IPN/GNPs, wesequenced DNA sample using the same matrix solution thatwas freshly prepared and had been stored at 47C for 5 and8 months, respectively. The sequencing resolutions of DNAsample did not show any obvious change with time, whichindicates that the quasi-IPN/GNPs matrix solutions were stillvery stable after several months and their shelf life was long.

4 Concluding remarks

GNPs were prepared and added into a quasi-IPN composedof LPA with lower molecular mass (3.3 MDa) and PDMA toform polymer/metal composite matrices for DNA sequenc-ing by CE. The studies of intrinsic viscosity and DSC onquasi-IPN and quasi-IPN/GNPs indicate that there wereinteractions between GNPs and polymer chains and physicalcross-linking points might be formed. The sequencingresults show that the resolutions of quasi-IPN/GNPs werehigher than those of quasi-IPN without GNPs and approxi-mated those of quasi-IPN-H with LPA (6.5 MDa) and with-out GNPs. However, the resolutions would decrease withincrease in the GNP concentration for larger DNA frag-ments, and the overall resolutions decreased with increasingtemperature. Moreover, the separation would be faster whena small amount of GNPs was added into sequencing matri-ces or temperature was increased. In other words, in thepresence of the GNPs, the separation was fast and efficient,and the resolution was high. The reproducibility of quasi-IPN/GNPs solution was excellent and its shelf life was morethan eight months.

Resolution is determined by two parameters: selectivityand separation efficiency. There was no significant change inselectivity but there was notable difference in separationefficiency with varied concentrations of GNPs and tempera-ture. Therefore, higher sieving ability can be explained as aresult of increase in separation efficiency. In the presence ofGNPs, the superior sequencing efficiency is possible due tothe interactions of GNPs with polymer chains, which pre-vented the polymer chains from sliding away from eachother and thus could form relatively more stable “pore” sizes(more robust sieving matrix networks), and increase theapparent molecular weight of the matrix and its sievingproperties, as also demonstrated by comparing the sequenc-ing performances of quasi-IPN/GNPs-1 with quasi-IPN-H

(with 6.5 MDa LPA and without GNPs). Moreover, high effi-ciency is partially due to minimized DNA adsorption on thecapillary wall in the presence of GNPs that were adsorbed onthe wall, leading to a decrease in EOF.

Optimization of variables, such as particle size of GNPsand electric field strength, will be performed in the future tofurther improve the sequencing performance. Furthermore,quasi-IPN/GNPs matrices containing LPA with a molecularmass lower than 3.3 MDa will be prepared and applied to thesequence in order to further investigate the influence ofGNPs on sieving performance.

We greatly acknowledge the support of this work by theNational Natural Science Foundation of China (grant no.50373040), the Scientific Research Foundation for the ReturnedOverseas Chinese Scholars, State Education Ministry, and theFoundation for Development of Talent of Anhui Province (grantno. 2005Z026).

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novel quasi-interpenetrating network/gold nanoparticles composite matrices for dna sequencing by ce

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