ICP-MS-Based Multiplex and Ultrasensitive Assay of Viruses with

Sep 27, 2013 - Highly sensitive and a multiplex assay of viruses and viral DNAs in complex biological samples is extremely important for clinical diag...
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ICP-MS-Based Multiplex and Ultrasensitive Assay of Viruses with Lanthanide-Coded Biospecific Tagging and Amplification Strategies Yacui 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 and Chemical 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 viruses and viral DNAs in complex biological samples is extremely important for clinical diagnosis and prognosis of pathogenic diseases as well as virology studies. We present an effective ICP-MS-based multiplex and ultrasensitive assay of viral DNAs with lanthanide-coded oligonucleotide hybridization and rolling circle amplification (RCA) strategies on biofunctional magnetic nanoparticles (MNPs), in which single-stranded capture DNA (ss-Cap-DNA)-functionalized MNPs (up to 1.65 × 104 ss-Cap-DNA per MNP) were used to recognize and enrich target DNAs, and single-stranded report DNA (ss-RepDNA-DOTA-Ln) coded by the lanthanide−DOTA complex hybridized with the targeted DNA for highly sensitive readout of HIV (28 amol), HAV (48 amol), and HBV (19 amol). When utilizing the RCA technique in association with the design and synthesis 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 this ICP-MS-based multiplex DNA assay for clinical use. One can expect that this element-coded ICP-MS-based multiplex and ultrasensitive DNA assay will play an ever more important role in the fields of bioanalysis and virology and in medical studies after further sophisticated modifications.

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methods such as electrochemical coding technology15,16 and surface-enhanced Raman scattering (SERS)17−21 have also been adapted to a multiplex DNA assay when the nanocrystals or the SERS reporters were carefully selected, but much care has to be paid in precisely controlling the incorporating agents during the fabrication of substrates and spectrum-resolved reporters. A novel alternative encoding strategy combining the use of a highly selective and sensitive tool is greatly desired to meet the demands for acquiring multiplex information concerning biomolecules from a complex biological system. Elemental mass spectrometry (especially, inductively-coupled plasma-mass spectrometry, ICP-MS) is a promising tool having multiplex analysis ability with mass-based individual element and/or isotope resolution. The resolution comes simply from clear distinguishing of the mass-to-charge ratio of the different element/isotope in a mass spectrum. In addition to the massbased resolution of ICP-MS, ICP (a very hard ionization source) results in the element/isotope MS signal being almost independent of the sample matrix when compared with a soft

uman pathogenic diseases, such as HIV/AIDS and viral hepatitis, have high infectivity but few effective treatments.1 Moreover, coinfection with these viruses is considered to be linked to higher mortality.2−4 Effective multiplex and ultrasensitive assay of these viruses for early diagnosis and/or prognosis is thus needed for subsequent early and efficacious therapy to improve the quality of life and life expectancy of patients. Besides the classical route for the diagnosis of viral infection via testing antibodies in serum, nucleic acid testing has significantly aided pathologists in the etiological diagnosis and management of a disease by quantifying the unique nucleic acid sequence of the virus and thus the virus itself.5 Among the methods developed for the multiplex detection of DNAs so far, the fluorescent-based platform is the most widely used approach.6−8 However, its inherent drawbacks as we already know, for example, photobleaching and especially broadspectrum emission, result in the platform frequently encountering spectral overlap to some extent, limiting their application in a high-level multiplex quantification. Even using quantum dots with their merits of broad absorption spectra and narrow sizetunable photoluminescence,9−12 the number of spectrally distinct codes that can be employed for encoding is very limited and far less than that theoretically predicted.13,14 Other © 2013 American Chemical Society

Received: August 2, 2013 Accepted: September 27, 2013 Published: September 27, 2013 9428

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Scheme 1. (A) ICP-MS-Based Multiplex DNA Assay with Lanthanide-Coded Oligonucleotide Hybridization Strategy and Biofunctional 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

Information), and then the corresponding biotinylated singlestranded capture DNA (ss-Cap-DNA) designed and synthesized (Table S2 of the Supporting Information) toward targeted DNA was anchored on the Fe3O4@SiO2−SA via the specific interaction between biotin and SA. The Fe3O4@SiO2− SA-(ss-Cap-DNA) obtained was then used to capture and enrich the unique DNA sequence of the targeted virus based on the principle of Watson−Crick base pairing and to magnetically separate them out of a complex biological medium (Scheme 1A). Subsequently, the remaining segment of the targeted DNA was hybridized with the designed and synthesized ss-report DNA covalently encoded with a complex of Ln-coordinated 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid (DOTALn) (ss-Rep-DNA-DOTA-Ln) (Table S2 of the Supporting Information and Scheme 1C) for element-resolved and sensitive readouts via determination of the nonradioactive Ln encoded using ICP-MS (Scheme 1A). Furthermore, considering the replication characteristic of DNA, we designed and synthesized a longer “bridge” DNA (Table S2 of the Supporting Information), consisting of a complementary DNA sequence for specific hybridization with the targeted DNA, and a primer sequence, which is complementary with ssRep-DNA-DOTA-Ln for rolling circle amplification (RCA). In this way, much more ss-Rep-DNA-DOTA-Ln can be attached to the repeated sequences of RCA products for a more sensitive readout 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 silica layer of 25 nm determined using TEM, as shown in Figures S1−S2 of the Supporting Information. Subsequently, the

ionization source (ESI and/or MALDI) mass spectrometry, offering more accurate and reliable information on element/ isotope absolute contents, which, in turn, reflects the concentrations of biomolecules. These characteristics facilitate the development of novel approaches with chemical selectivity and/or biospecificity toward the biomolecules using only one of any 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 demonstrate their superior advantage and application potential over the reported optical methods regarding the spectral overlapping problem.25−36 Although a few element/isotope-encoding strategies combined with ICP-MS detection have been applied to sensitive and multiplex DNA assay at the picomolar level,37−40 we hypothesize that oligonucleotide hybridizationbased biospecific element/isotope-encoding strategy together with rolling circle amplification on biofunctional magnetic nanoparticles using ICP-MS will demonstrate a fairly multiplex assay of DNAs and be able to further improve the limit of detection of viral DNA. We report an ICP-MS-based multiplex and ultrasensitive assay of viruses with lanthanide (Ln)-coded biospecific tagging and amplification strategies on biofunctional magnetic nanoparticles via determination of the Ln encoded to their unique DNA sequences using ICP-MS (Table S1 of the Supporting Information) in this proof-of-concept study (Scheme 1, panels A−D). Silica-coated Fe3O4-based magnetic nanoparticles (Fe3O4@SiO2-MNPs) were used to covalently immobilize streptavidin (SA) via carbodiimide chemistry to obtain Fe3O4@ SiO2−SA (Scheme 1B and Figures S1−S5 in the Supporting 9429

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HAV (2), HBV (3), (HIV + HAV) (4), (HIV + HBV) (5), (HAV + HBV) (6), (HIV + HAV + HBV) (7), and a blank sample were prepared, and then all three types of Fe3O4@ SiO2−SA-(ss-Cap-DNA) (50 μg) and corresponding ss-RepDNA-DOTA-Ln (Ln = Eu3+, Tb3+, and Ho3+) (200 pmol, 10fold the amounts of the targeted DNAs in the samples) were added and hybridized at 25 °C for 30 min. After the procedures of magnetic separation through washing and resuspension and dehybridization, the ss-Rep-DNA-DOTA-Ln in the supernatants were analyzed using ICP-MS. As can be seen in Figure 1A, no cross-hybridization signals were observed, demonstrat-

surface of the Fe3O4@SiO2-MNPs was modified with amino groups using 3-aminopropyltriethoxysiane in ethanol (Fe3O4@ SiO2−NH2)43 and then carboxyl-functionalization through the reaction between the succinic anhydride and the amine modified on the Fe3O4@SiO2-MNPs in anhydrous DMF (Figure S3 and S4 of the Supporting Information). The uniform core/shell carboxyl-functionalized Fe3O4@SiO2-MNPs (Fe3O4@SiO2−COOH) obtained were further characterized using DLS. The ξ potential of the Fe3O4@SiO2−COOH prepared was −29.37 ± 1.62 mV, and those of the Fe3O4@SiO2 and Fe3O4@SiO2−NH2 were −20.13 ± 2.55 and 33.03 ± 0.58 mV, suggesting the successful modification of the −NH2 and then −COOH groups. In order to anchor the biotinylated ssCap-DNA (Scheme 1B) onto the Fe3O4@SiO2−COOH, SA was bonded covalently on the Fe3O4@SiO2−COOH via the reaction between −COOH and the −NH2 from the lysine residues of SA to obtain Fe3O4@SiO2−SA. The biotinylated ssCap-DNA (Table S2 of the Supporting Information) was thus attached to the surface of the Fe3O4@SiO2−SA via the interaction between SA and biotin (Scheme 1B). The resultant Fe3O4@SiO2−SA-(ss-Cap-DNA) MNPs were washed with a washing buffer (W-buffer) and further blocked with a blocking buffer containing 1% BSA in the hybridization buffer (H-buffer) to reduce the undesired nonspecific absorption sites on the surface (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 proofof-concept study, we designed and synthesized three kinds of ss-Rep-DNA-DOTA-Ln (Ln = Eu 3+ , Tb 3+ , and Ho 3+ , respectively) (Scheme 1C and Table S2 of the Supporting Information), complementary to the unique DNA sequences of HIV, HAV, and HBV. Successful conjugation of maleimidoethylacetamide DOTA-Ln to the ss-Rep-DNA via the reaction between 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 of ss-Cap-DNA anchored on the surface of the Fe3O4@SiO2−SA was thus determined using ICP-MS via experiments through adding various amounts of the target DNA (from 1 to 500 nM) and 200 pmol ss-Rep-DNA-DOTA-Ln (Scheme 1A and Figure S7 of the Supporting Information) into the solution containing 50 μg Fe3O4@SiO2−SA-(ss-Cap-DNA) (7.29 × 108 particles, see the Supporting Information). After hybridization in the Hbuffer, 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 and then washing with the W-buffer three times and then the Hbuffer twice, in sequence. The Fe3O4@SiO2−SA-(ss-CapDNA)/(target DNA)/(ss-Rep-DNA-DOTA-Ln) obtained were resuspended and dehybridized by heating at 90 °C for 20 min, and the released ss-Rep-DNA-DOTA-Ln were collected for subsequent ICP-MS analysis (Scheme 1A). The number of ss-Rep-DNA-DOTA-Ln labeled was thus calculated to be 1.65 × 104 per particle (see the Supporting Information), which is equal to that of the ss-Cap-DNA, according to the 1to-1 base complementary pairing principle. These results suggested that more than 4 orders of magnitude-targeted DNA could be enriched on the MNPs, resulting in at least 4 orders of magnitude improvement in sensitivity when compared with just using the ss-Cap-DNA and ss-Rep-DNADOTA-Ln in a homogeneous solution. To demonstrate our proposal, seven artificial samples (Table S3 of the Supporting Information) containing HIV (sample 1),

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

ing that the method we developed was selective and suitable for multiplex assay of the targeted DNAs. Moreover, a series of solutions containing the unique DNA sequences of HIV, HAV, and HBV from 10 pM (no concentration lower than 10 pM was tested) to 500 nM were employed to test calibration linearity. Figure 1 (panels B−D) indicates that the MS signal intensities of Eu3+, Tb3+, and Ho3+, corresponding to HIV, HAV, and HBV, increased linearly along with the increase in the concentrations of the target DNAs in the range from 10 pM to 100 nM (Figure 1, panels B−D), with correlation coefficients of 0.9963, 0.9934, and 0.9998, respectively. Increasing viral DNA concentration beyond 100 nM resulted in a nonlinear MS response (data not shown). Under these conditions, the method detection limits (MDL, 3σ) were determined to be 28 amol HIV, 48 amol HAV, and 19 amol HBV, respectively, and their RSDs were 3.6, 2.4, and 4.7% at 10 nM (n = 6). Next, we applied this method to determine HIV, HAV, and HBV in 4T1 cell lysates and serum (Figure S8 of the Supporting Information). The results obtained (Table S4 of the Supporting Information) indicated that the recoveries were between 94.2 to 109.1%. 9430

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hybridization of DNA allow an excellent multiplex assay of DNAs and thus the corresponding viruses without the aid of any chromatography and/or electrophoresis-based techniques. Further development of the ICP-MS-based multiplex DNA assay can be expected to design a novel circularized DNA template, which contains more specific regions, for simultaneously labeling the differently repeated sequences of the RCA product with Ln and isotope-enriched Ln to perform accurate isotope dilution quantification of DNA and thus the virus itself in the near future. This research is currently ongoing in our laboratory.

The detection ability of the above strategy could be further improved when utilizing the replication property of DNA. We took HBV as an example here and then designed and synthesized a longer “bridge” DNA (Scheme 1D and Table S2 of the Supporting Information). It contains a segment of 15 base pairs (bp) for recognizing the unique DNA sequence of HBV, and a spacer of 10 thymidine nucleotides, as well as a primer sequence of 20 bp for RCA (Table S2 of the Supporting Information).44,45 A circularized DNA template (50 bp) (Scheme 1D) containing a 20 bp region that was complementary to the primer sequence was designed and synthesized (Table S2 and Figure S9 of the Supporting Information). A corresponding ss-Rep-DNA-DOTA-Ho (Table S2 of the Supporting Information), which has an identical sequence to the 20 bp region in the circle template DNA, was thus prepared for subsequently labeling the RCA products. The RCA was initiated by adding phi29 DNA polymerase in the presence of dNTPs, and a long single stranded DNA was thus generated containing hundreds of replicates of the 20 bp primer DNA sequence after the optimized 2 h amplification (see the Supporting Information). The repeated sequences of RCA products were then hybridized with the corresponding ss-RepDNA-DOTA-Ho (Scheme 1D). As high as 190-fold 165Ho signal enhancement was observed when compared with that without RCA, suggesting that 190 rounds were performed after 2 h of amplification and showed a linear dynamic range from 250 fM (no concentration lower than 250 fM was tested) to 1 nM with a linear correlation coefficient of 0.9905 (Figure 2A)

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details and additional characterization data. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86 (0)592 2187400. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the National Natural Science Foundation of China (Grants 21035006 and 21275120) and the National Basic Research 973 Project (Grant 2014CB932004). We thank Prof. John Hodgkiss of The University of Hong Kong for helping with the English in this article.

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Figure 2. (A) Calibration curve of HBV with the RCA-based signal enhancement method. (B) Comparison of the ICP-MS signals of Howith Ln-coded oligonucleotide hybridization (black) and RCA (red) strategies. Error bars (SD) were calculated from three independent experiments.

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