ICP-MS-Based Multiplex and Ultrasensitive Assay of Viruses withLanthanide-Coded Biospecific Tagging and Amplification StrategiesYacui Luo,† Xiaowen Yan,† Yishun Huang,† Ruibin Wen,† Zhaoxin Li,† Limin Yang,†

Chaoyong James Yang,† and Qiuquan Wang*,†,‡

†Department of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry andChemical Engineering, Xiamen University, Xiamen 361005, China‡State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, China

*S Supporting Information

ABSTRACT: Highly sensitive and a multiplex assay of virusesand viral DNAs in complex biological samples is extremelyimportant for clinical diagnosis and prognosis of pathogenicdiseases as well as virology studies. We present an effectiveICP-MS-based multiplex and ultrasensitive assay of viral DNAswith lanthanide-coded oligonucleotide hybridization androlling circle amplification (RCA) strategies on biofunctionalmagnetic nanoparticles (MNPs), in which single-strandedcapture DNA (ss-Cap-DNA)-functionalized MNPs (up to 1.65× 104 ss-Cap-DNA per MNP) were used to recognize andenrich target DNAs, and single-stranded report DNA (ss-Rep-DNA-DOTA-Ln) coded by the lanthanide−DOTA complexhybridized with the targeted DNA for highly sensitive readoutof HIV (28 amol), HAV (48 amol), and HBV (19 amol). When utilizing the RCA technique in association with the design andsynthesis of a “bridge” DNA and a corresponding ss-Rep-DNA-DOTA-Ho, as low as 90 zmol HBV could be detected.Preliminary applications to the determination of the viral DNAs in 4T1 cell lysates and in serum confirmed the feasibility of thisICP-MS-based multiplex DNA assay for clinical use. One can expect that this element-coded ICP-MS-based multiplex andultrasensitive DNA assay will play an ever more important role in the fields of bioanalysis and virology and in medical studiesafter further sophisticated modifications.

Human pathogenic diseases, such as HIV/AIDS and viralhepatitis, have high infectivity but few effective treat-

ments.1 Moreover, coinfection with these viruses is consideredto be linked to higher mortality.2−4 Effective multiplex andultrasensitive assay of these viruses for early diagnosis and/orprognosis is thus needed for subsequent early and efficacioustherapy to improve the quality of life and life expectancy ofpatients. Besides the classical route for the diagnosis of viralinfection via testing antibodies in serum, nucleic acid testing hassignificantly aided pathologists in the etiological diagnosis andmanagement of a disease by quantifying the unique nucleic acidsequence of the virus and thus the virus itself.5 Among themethods developed for the multiplex detection of DNAs so far,the fluorescent-based platform is the most widely usedapproach.6−8 However, its inherent drawbacks as we alreadyknow, for example, photobleaching and especially broad-spectrum emission, result in the platform frequently encounter-ing spectral overlap to some extent, limiting their application ina high-level multiplex quantification. Even using quantum dotswith their merits of broad absorption spectra and narrow size-tunable photoluminescence,9−12 the number of spectrallydistinct codes that can be employed for encoding is verylimited and far less than that theoretically predicted.13,14 Other

methods such as electrochemical coding technology15,16 andsurface-enhanced Raman scattering (SERS)17−21 have also beenadapted to a multiplex DNA assay when the nanocrystals or theSERS reporters were carefully selected, but much care has to bepaid in precisely controlling the incorporating agents during thefabrication of substrates and spectrum-resolved reporters. Anovel alternative encoding strategy combining the use of ahighly selective and sensitive tool is greatly desired to meet thedemands for acquiring multiplex information concerningbiomolecules from a complex biological system.Elemental mass spectrometry (especially, inductively-coupled

plasma-mass spectrometry, ICP-MS) is a promising tool havingmultiplex analysis ability with mass-based individual elementand/or isotope resolution. The resolution comes simply fromclear distinguishing of the mass-to-charge ratio of the differentelement/isotope in a mass spectrum. In addition to the mass-based resolution of ICP-MS, ICP (a very hard ionizationsource) results in the element/isotope MS signal being almostindependent of the sample matrix when compared with a soft

Received: August 2, 2013Accepted: September 27, 2013Published: September 27, 2013

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ionization source (ESI and/or MALDI) mass spectrometry,offering more accurate and reliable information on element/isotope absolute contents, which, in turn, reflects theconcentrations of biomolecules. These characteristics facilitatethe development of novel approaches with chemical selectivityand/or biospecificity toward the biomolecules using only one ofany known species of the element or isotopic standards.22−24

Recent studies on ICP-MS-based multiplex assays of protein/peptides with element/isotope-encoding strategies demonstratetheir superior advantage and application potential over thereported optical methods regarding the spectral overlappingproblem.25−36 Although a few element/isotope-encodingstrategies combined with ICP-MS detection have been appliedto sensitive and multiplex DNA assay at the picomolarlevel,37−40 we hypothesize that oligonucleotide hybridization-based biospecific element/isotope-encoding strategy togetherwith rolling circle amplification on biofunctional magneticnanoparticles using ICP-MS will demonstrate a fairly multiplexassay of DNAs and be able to further improve the limit ofdetection of viral DNA.We report an ICP-MS-based multiplex and ultrasensitive

assay of viruses with lanthanide (Ln)-coded biospecific taggingand amplification strategies on biofunctional magnetic nano-particles via determination of the Ln encoded to their uniqueDNA sequences using ICP-MS (Table S1 of the SupportingInformation) in this proof-of-concept study (Scheme 1, panelsA−D). Silica-coated Fe3O4-based magnetic nanoparticles(Fe3O4@SiO2-MNPs) were used to covalently immobilizestreptavidin (SA) via carbodiimide chemistry to obtain Fe3O4@SiO2−SA (Scheme 1B and Figures S1−S5 in the Supporting

Information), and then the corresponding biotinylated single-stranded capture DNA (ss-Cap-DNA) designed and synthe-sized (Table S2 of the Supporting Information) towardtargeted DNA was anchored on the Fe3O4@SiO2−SA via thespecific interaction between biotin and SA. The Fe3O4@SiO2−SA-(ss-Cap-DNA) obtained was then used to capture andenrich the unique DNA sequence of the targeted virus based onthe principle of Watson−Crick base pairing and to magneticallyseparate them out of a complex biological medium (Scheme1A). Subsequently, the remaining segment of the targeted DNAwas hybridized with the designed and synthesized ss-reportDNA covalently encoded with a complex of Ln-coordinated1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid (DOTA-Ln) (ss-Rep-DNA-DOTA-Ln) (Table S2 of the SupportingInformation and Scheme 1C) for element-resolved andsensitive readouts via determination of the nonradioactive Lnencoded using ICP-MS (Scheme 1A). Furthermore, consider-ing the replication characteristic of DNA, we designed andsynthesized a longer “bridge” DNA (Table S2 of theSupporting Information), consisting of a complementaryDNA sequence for specific hybridization with the targetedDNA, and a primer sequence, which is complementary with ss-Rep-DNA-DOTA-Ln for rolling circle amplification (RCA). Inthis way, much more ss-Rep-DNA-DOTA-Ln can be attachedto the repeated sequences of RCA products for a more sensitivereadout of the targeted DNA using ICP-MS (Scheme 1D).First, Fe3O4@SiO2-MNPs were synthesized according to the

reported methods.41,42 The overall size was 320 nm with a silicalayer of 25 nm determined using TEM, as shown in FiguresS1−S2 of the Supporting Information. Subsequently, the

Scheme 1. (A) ICP-MS-Based Multiplex DNA Assay with Lanthanide-Coded Oligonucleotide Hybridization Strategy andBiofunctional Magnetic Nanoparticle (MNP) Enrichment. (B) Fabrication of the Fe3O4@SiO2−SA-ss-Cap-DNA MNPs. (C)Preparation of Ln-Coded ss-Rep-DNA. (D) Solid Phase RCA for Signal Amplification of ICP-MS-Based DNA Assay

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surface of the Fe3O4@SiO2-MNPs was modified with aminogroups using 3-aminopropyltriethoxysiane in ethanol (Fe3O4@SiO2−NH2)

43 and then carboxyl-functionalization through thereaction between the succinic anhydride and the aminemodified on the Fe3O4@SiO2-MNPs in anhydrous DMF(Figure S3 and S4 of the Supporting Information). Theuniform core/shell carboxyl-functionalized Fe3O4@SiO2-MNPs(Fe3O4@SiO2−COOH) obtained were further characterizedusing DLS. The ξ potential of the Fe3O4@SiO2−COOHprepared was −29.37 ± 1.62 mV, and those of the Fe3O4@SiO2and Fe3O4@SiO2−NH2 were −20.13 ± 2.55 and 33.03 ± 0.58mV, suggesting the successful modification of the −NH2 andthen −COOH groups. In order to anchor the biotinylated ss-Cap-DNA (Scheme 1B) onto the Fe3O4@SiO2−COOH, SAwas bonded covalently on the Fe3O4@SiO2−COOH via thereaction between −COOH and the −NH2 from the lysineresidues of SA to obtain Fe3O4@SiO2−SA. The biotinylated ss-Cap-DNA (Table S2 of the Supporting Information) was thusattached to the surface of the Fe3O4@SiO2−SA via theinteraction between SA and biotin (Scheme 1B). The resultantFe3O4@SiO2−SA-(ss-Cap-DNA) MNPs were washed with awashing buffer (W-buffer) and further blocked with a blockingbuffer containing 1% BSA in the hybridization buffer (H-buffer)to reduce the undesired nonspecific absorption sites on thesurface (see the Supporting Information).On the basis of our previous studies,31,33−35 Ln can be loaded

into the DOTA moiety to achieve Ln encoding. In this proof-of-concept study, we designed and synthesized three kinds ofss-Rep-DNA-DOTA-Ln (Ln = Eu3+, Tb3+, and Ho3+,respectively) (Scheme 1C and Table S2 of the SupportingInformation), complementary to the unique DNA sequences ofHIV, HAV, and HBV. Successful conjugation of maleimidoe-thylacetamide DOTA-Ln to the ss-Rep-DNA via the reactionbetween the maleimide group and the thiol (−SH) at the 3′end of ss-Rep-DNA was confirmed using MALDI-TOF-MS(see Figure S6 of the Supporting Information). The number ofss-Cap-DNA anchored on the surface of the Fe3O4@SiO2−SAwas thus determined using ICP-MS via experiments throughadding various amounts of the target DNA (from 1 to 500 nM)and 200 pmol ss-Rep-DNA-DOTA-Ln (Scheme 1A and FigureS7 of the Supporting Information) into the solution containing50 μg Fe3O4@SiO2−SA-(ss-Cap-DNA) (7.29 × 108 particles,see the Supporting Information). After hybridization in the H-buffer, the hybridization complex obtained, Fe3O4@SiO2−SA-(ss-Cap-DNA)/(targeted DNA)/(ss-Rep-DNA-DOTA-Ln),was separated by applying an external magnetic field andthen washing with the W-buffer three times and then the H-buffer twice, in sequence. The Fe3O4@SiO2−SA-(ss-Cap-DNA)/(target DNA)/(ss-Rep-DNA-DOTA-Ln) obtainedwere resuspended and dehybridized by heating at 90 °C for20 min, and the released ss-Rep-DNA-DOTA-Ln werecollected for subsequent ICP-MS analysis (Scheme 1A). Thenumber of ss-Rep-DNA-DOTA-Ln labeled was thus calculatedto be 1.65 × 104 per particle (see the Supporting Information),which is equal to that of the ss-Cap-DNA, according to the 1-to-1 base complementary pairing principle. These resultssuggested that more than 4 orders of magnitude-targetedDNA could be enriched on the MNPs, resulting in at least 4orders of magnitude improvement in sensitivity whencompared with just using the ss-Cap-DNA and ss-Rep-DNA-DOTA-Ln in a homogeneous solution.To demonstrate our proposal, seven artificial samples (Table

S3 of the Supporting Information) containing HIV (sample 1),

HAV (2), HBV (3), (HIV + HAV) (4), (HIV + HBV) (5),(HAV + HBV) (6), (HIV + HAV + HBV) (7), and a blanksample were prepared, and then all three types of Fe3O4@SiO2−SA-(ss-Cap-DNA) (50 μg) and corresponding ss-Rep-DNA-DOTA-Ln (Ln = Eu3+, Tb3+, and Ho3+) (200 pmol, 10-fold the amounts of the targeted DNAs in the samples) wereadded and hybridized at 25 °C for 30 min. After the proceduresof magnetic separation through washing and resuspension anddehybridization, the ss-Rep-DNA-DOTA-Ln in the super-natants were analyzed using ICP-MS. As can be seen in Figure1A, no cross-hybridization signals were observed, demonstrat-

ing that the method we developed was selective and suitable formultiplex assay of the targeted DNAs. Moreover, a series ofsolutions containing the unique DNA sequences of HIV, HAV,and HBV from 10 pM (no concentration lower than 10 pM wastested) to 500 nM were employed to test calibration linearity.Figure 1 (panels B−D) indicates that the MS signal intensitiesof Eu3+, Tb3+, and Ho3+, corresponding to HIV, HAV, andHBV, increased linearly along with the increase in theconcentrations of the target DNAs in the range from 10 pMto 100 nM (Figure 1, panels B−D), with correlation coefficientsof 0.9963, 0.9934, and 0.9998, respectively. Increasing viralDNA concentration beyond 100 nM resulted in a nonlinear MSresponse (data not shown). Under these conditions, themethod detection limits (MDL, 3σ) were determined to be 28amol HIV, 48 amol HAV, and 19 amol HBV, respectively, andtheir RSDs were 3.6, 2.4, and 4.7% at 10 nM (n = 6). Next, weapplied this method to determine HIV, HAV, and HBV in 4T1cell lysates and serum (Figure S8 of the SupportingInformation). The results obtained (Table S4 of the SupportingInformation) indicated that the recoveries were between 94.2to 109.1%.

Figure 1. (A) Selectivity of the developed ICP-MS-based multiplexDNA assay in seven different samples. Samples 1−3: only one virusDNA was present in the sample (HIV, HAV, and HBV, respectively);samples 4−6: two viral DNAs were present (HIV and HAV, HIV andHBV, HAV and HBV); sample 7: all three viral DNAs were present.(B−D) Standard calibration curves of HIV, HAV, and HBV in the H-buffer with Ln-coded oligonucleotide hybridization strategy using ICP-MS. Error bars (SD) were calculated from three independentexperiments.

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The detection ability of the above strategy could be furtherimproved when utilizing the replication property of DNA. Wetook HBV as an example here and then designed andsynthesized a longer “bridge” DNA (Scheme 1D and TableS2 of the Supporting Information). It contains a segment of 15base pairs (bp) for recognizing the unique DNA sequence ofHBV, and a spacer of 10 thymidine nucleotides, as well as aprimer sequence of 20 bp for RCA (Table S2 of the SupportingInformation).44,45 A circularized DNA template (50 bp)(Scheme 1D) containing a 20 bp region that was comple-mentary to the primer sequence was designed and synthesized(Table S2 and Figure S9 of the Supporting Information). Acorresponding ss-Rep-DNA-DOTA-Ho (Table S2 of theSupporting Information), which has an identical sequence tothe 20 bp region in the circle template DNA, was thus preparedfor subsequently labeling the RCA products. The RCA wasinitiated by adding phi29 DNA polymerase in the presence ofdNTPs, and a long single stranded DNA was thus generatedcontaining hundreds of replicates of the 20 bp primer DNAsequence after the optimized 2 h amplification (see theSupporting Information). The repeated sequences of RCAproducts were then hybridized with the corresponding ss-Rep-DNA-DOTA-Ho (Scheme 1D). As high as 190-fold 165Hosignal enhancement was observed when compared with thatwithout RCA, suggesting that 190 rounds were performed after2 h of amplification and showed a linear dynamic range from250 fM (no concentration lower than 250 fM was tested) to 1nM with a linear correlation coefficient of 0.9905 (Figure 2A)

when plotting the logarithm of ICP-MS intensity versus HBVconcentration. Under this condition, the MDL (3σ) for HBVwas determined to be 90 zmol with an RSD of 5.9% (n = 6) at1 pM. Further application to determine HBV in three 4T1 celllysates and three serums samples indicated that the averagerecoveries were 110 and 105% (Table S4 of the SupportingInformation). When compared with the nonRCA methoddeveloped, we could gain more than 2 orders of magnitudeimprovement in sensitivity (Figure 2B).In conclusion, we developed an ICP-MS-based multiplex

assay of pathogenic viruses via the determination of Lnencoding of their unique DNAs, using oligonucleotidehybridization strategy. Down to 90 zmol of HBV could bedetermined when utilizing the RCA technique in addition tothe contribution from the thousands of ss-Cap-DNAs anchoredon the surface of the biofunctional MNPs and the highlysensitive detection of Ln by ICP-MS. Mass-based element/isotope resolution of ICP-MS and specific base pairing

hybridization of DNA allow an excellent multiplex assay ofDNAs and thus the corresponding viruses without the aid ofany chromatography and/or electrophoresis-based techniques.Further development of the ICP-MS-based multiplex DNAassay can be expected to design a novel circularized DNAtemplate, which contains more specific regions, for simulta-neously labeling the differently repeated sequences of the RCAproduct with Ln and isotope-enriched Ln to perform accurateisotope dilution quantification of DNA and thus the virus itselfin the near future. This research is currently ongoing in ourlaboratory.

■ ASSOCIATED CONTENT*S Supporting InformationExperimental details and additional characterization data. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: qqwang@xmu.edu.cn. Fax: +86 (0)592 2187400.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis study was financially supported by the National NaturalScience Foundation of China (Grants 21035006 and21275120) and the National Basic Research 973 Project(Grant 2014CB932004). We thank Prof. John Hodgkiss of TheUniversity of Hong Kong for helping with the English in thisarticle.

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