Multiplex Detection of DNA Sequences Using the Volume-Amplified

Anal. Chem. 2009, 81, 3398–3406

Multiplex Detection of DNA Sequences Using the Volume-Amplified Magnetic Nanobead Detection Assay Mattias Stro¨mberg,† Teresa Zarda´n Go´mez de la Torre,† Jenny Go¨ransson,§ Klas Gunnarsson,‡ Mats Nilsson,*,§ Peter Svedlindh,*,‡ and Maria Strømme*,† Department of Engineering Sciences, Division of Nanotechnology and Functional Materials, Uppsala University, The Ångstro¨m Laboratory, Box 534, SE-751 21 Uppsala, Sweden, Department of Engineering Sciences, Division of Solid State Physics, Uppsala University, The Ångstro¨m Laboratory, Box 534, SE-751 21 Uppsala, Sweden, and Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85 Uppsala, Sweden The possibility for conducting multiplex detection of DNAsequences using the volume-amplified magnetic nanobead detection assay [Stro ¨mberg, M.; Go ¨ransson, J.; Gunnarsson, K.; Nilsson, M.; Svedlindh, P.; Strømme, M. Nano Lett. 2008, 8, 816-821] was investigated. In this methodology, a batch consisting of a mixture of several sizes of probe-tagged magnetic beads was used for detection of several types of targets in the same compartment. Furthermore, a nonlinear least-squares deconvolution procedure of the composite imaginary part of complex magnetization vs frequency spectra based on the Cole-Cole model was applied to analyze the data. The results of a quantitative biplex analysis experiment were compared with the corresponding separate single-target assays. Finally, triplex analysis was briefly demonstrated qualitatively. Biplex and triplex detection were found to perform well qualitatively. Biplex detection was found to enable a rough target quantification. Multiplex detection may become a complement to performing multiple separate single-target assays for, e.g., parallel detection of multiple infectious pathogens. Multiplex detection also permits robust relative quantification and inclusion of an internal control to improve quantification accuracy. Today, there is an increasing interest to develop new nanotechnologies for clinical diagnostic applications, i.e., nanodiagnostic tools or nanobiosensors.1,2 These should exhibit high sensitivity, specificity, and cost-effectiveness but also allow for multiplex analysis in order to meet the future needs from various fields such as in vitro medical diagnostics, pharmaceutical discovery, and pathogen detection.1,2 In particular, detection of pathogenic bacteria is of great importance for prevention and * To whom correspondence should be addressed. E-mail: mats.nilsson@ genpat.uu.se (M.N.); [email protected] (P.S.); maria.stromme@ angstrom.uu.se (M.S.). Phone: +46 18 471 0000. † Department of Engineering Sciences, Division of Nanotechnology and Functional Materials. ‡ Department of Engineering Sciences, Division of Solid State Physics. § Department of Genetics and Pathology. (1) Azzazy, H. M. E.; Mansour, M. M. H.; Kazmierczak, S. C. Clin. Chem. 2006, 52, 1238–1246. (2) Erickson, D.; Mandal, S.; Yang, A. H. J.; Cordovez, B. Microfluid. Nanofluid. 2008, 4, 33–52.

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identification of health and safety problems.3 The former could for example be related to infectious diseases while the latter is of importance for, e.g., the food industry and to probe water and environment quality.3 Also, during recent years, the interest for developing DNA biosensors for diagnostic testing has grown rapidly.4 Numerous biosensor platforms and nanodiagnostic strategies currently exist such as fluorescence-based DNA microarrays,5,6 electrochemical nanobiosensors,7,8 acoustic sensors,9,10 cantilever biosensors,11 and various kinds of optical biosensors.12 Besides these bioassay technologies, the use of magnetic micro- or nanoparticles (beads) in biosensing offer a number of advantages since there is no significant magnetic background in most samples of interest.13 Furthermore, magnetic bead labels can be detected by relatively noninvasive methods, and they have high physical and chemical stability and are inexpensive to produce.13 Magnetic biosensing principles are roughly classified as being either substrate-based or substrate-free. Giant magnetoresistance biosensors14 and micro-Hall devices15 belong to the former category while the Brownian relaxation biosensor method belongs to the latter. The Brownian relaxation biosensor method was theoretically outlined by Connolly and St. Pierre16 and later experimentally (3) Lazcka, O.; Campo, F. J. D.; Mun ˜oz, F. X. Biosens. Bioelectron. 2007, 22, 1205–1217. (4) Sassolas, A.; Leca-Bouvier, B. D.; Blum, L. J. Chem. Rev. 2008, 108, 109– 139. (5) Park, H. G.; Song, J. Y.; Park, K. H.; Kim, M. H. Chem. Eng. Sci. 2006, 61, 954–965. (6) Petrik, J. Transfusion Med. 2006, 16, 233–247. (7) Wang, J. Biosens. Bioelectron. 2006, 21, 1887–1892. (8) Pumera, M.; Sa´nchez, S.; Ichinose, I.; Tang, J. Sens. Actuators, B 2007, 123, 1195–1205. (9) Gronewold, T. M. A. Anal. Chim. Acta 2007, 603, 119–128. ˇ iplys, D. Phys. Status Solidi A 2007, 204, (10) Chivukula, V. S.; Shur, M. S.; C 3209–3236. (11) Fritz, J. Analyst 2008, 133, 855–863. (12) Fan, X.; White, I. M.; Shopova, S. I.; Zhu, H.; Suter, J. D.; Sun, Y. Anal. Chim. Acta 2008, 620, 8–26. (13) Tamanaha, C. R.; Mulvaney, S. P.; Rife, J. C.; Whitman, L. J. Biosens. Bioelectron. 2008, 24, 1–13. (14) Wang, S. X.; Li, G. IEEE Trans. Magn. 2008, 44, 1687–1702. (15) Mihajlovic´, G. Ph.D. Dissertation, The Florida State University, Tallahassee, FL, 2006. (16) Connolly, J.; St. Pierre, T. G. J. Magn. Magn. Mater. 2001, 225, 156–160. 10.1021/ac900561r CCC: $40.75  2009 American Chemical Society Published on Web 03/31/2009

demonstrated by Astalan et al.17 and Hong et al.18 In this bioassay principle, probe biomolecules are coupled to suspended magnetic beads exhibiting Brownian relaxation behavior. Binding of target molecules to the bead surface causes a hydrodynamic size increase and thereby a decreased Brownian relaxation frequency of the beads. Recently, we demonstrated a new substrate-free sensitive (low picomolar range) and highly specific magnetic biosensor principle suitable as a potential platform for future development of lowcost diagnostic point-of-care and/or over-the-counter devices, the volume-amplified magnetic nanobead detection assay (VAM-NDA).19,20 To summarize the detection procedure of one single type of target-DNA, the assay begins with padlock probe target recognition21,22 followed by a very specific ligation reaction. Rolling circle amplification (RCA)23,24 of the circularized probetarget complexes for a certain time gives an ensemble of macromolecular DNA-coils (RCA-coils), which in turn are detected by letting detection probe oligonucleotide tagged magnetic nanobeads of one single size bind to the RCA-coils by base-pair hybridization (bead immobilization). This causes a strong decrease of the bead Brownian relaxation frequency (fB) due to the strongly increased bead hydrodynamic volume, now essentially corresponding to the size of an RCA-coil. Upon immobilization, the magnitude of the imaginary part (m′′) of the frequencydependent complex magnetization (m(f) ) m′(f) - im′′(f)) at the Brownian relaxation frequency for nonimmobilized beads, the high-frequency peak (HFP) level, decreases, compared to a negative control sample (absence of RCA-coils). RCA-coils containing immobilized beads (strictly speaking single RCAcoils and/or clusters of RCA-coils bound together by beads) are associated with a relaxation process, which we denote the low-frequency peak (LFP), and the magnitude of m′′ at the Brownian relaxation frequency associated with the LFP is denoted the LFP level. So far, we have demonstrated quantitative analysis of a single type of target-DNA19,20 and biplex detection of bacterial DNAsequences qualitatively.20 In the current work, we will in detail evaluate the possibility to conduct quantitative detection of two kinds of bacterial DNA-sequences simultaneously in the same compartment using sequences from Vibrio vulnificus and V. cholerae, i.e., quantitative biplex detection. The results from the biplex detection experiment will be compared with the corresponding single-target experiments conducted separately. We will also demonstrate qualitative triplex detection of bacterial DNA(17) Astalan, A. P.; Ahrentorp, F.; Johansson, C.; Larsson, K.; Krozer, A. Biosens. Bioelectron. 2004, 19, 945–951. (18) Hong, C.-Y.; Chen, W. S.; Jian, Z. F.; Yang, S. Y.; Horng, H. E.; Yang, L. C.; Yang, H. C. Appl. Phys. Lett. 2007, 90, 074105. (19) Stro ¨mberg, M.; Go ¨ransson, J.; Gunnarsson, K.; Nilsson, M.; Svedlindh, P.; Strømme, M. Nano Lett. 2008, 8, 816–821. (20) Stro ¨mberg, M.; Zarda´n Go´mez de la Torre, T.; Go ¨ransson, J.; Gunnarsson, K.; Nilsson, M.; Strømme, M.; Svedlindh, P. Biosens. Bioelectron. 2008, 24, 696–703. (21) Nilsson, M.; Malmgren, H.; Samiotaki, M.; Kwiatkowski, M.; Chowdhary, B. P.; Landegren, U. Science 1994, 265, 2085–2088. (22) Landegren, U.; Dahl, F.; Nilsson, M.; Fredriksson, S.; Bane´r, J.; Gullberg, M.; Jarvius, J.; Gustafsdottir, S.; So ¨derberg, O.; Ericsson, O.; Stenberg, J.; Schallmeiner, E. Comp. Funct. Genomics 2003, 4, 525–530. (23) Fire, A.; Xu, S.-Q. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4641–4645. (24) Liu, D.; Daubendiek, S. L.; Zillman, M. A.; Ryan, K.; Kool, E. T. J. Am. Chem. Soc. 1996, 118, 1587–1594.

sequences using sequences from V. vulnificus, V. cholerae, and Escherichia coli. THEORY Dynamic Magnetic Properties of Ferrofluids. The magnetic beads used in this paper are composed of a clustered core consisting of single domain maghemite nanoparticles, each having a diameter of ∼15 nm, held together by a dextran casing, where the nanoparticle magnetic moments are thermally blocked at room temperature and the beads are suspended in deionized water. With only one relaxation time present in the system, the Debye theory25 gives an expression for the complex low-field magnetization m(ω) for an ensemble of beads according to m(ω) ) (m0 - m∞)/(1 + iωτ) + m∞

(1)

where ω is the angular frequency of the applied ac magnetic excitation field, m∞ is the high-frequency magnetization, m0 is the low-field static magnetization, and τ is the characteristic relaxation time of the beads. The magnetic bead relaxation is governed by either of two relaxation mechanisms: The Ne´el relaxation,26 where the magnetic moment rotates within the nanoparticles, or the Brownian relaxation,27 where the entire bead rotates in response to the ac magnetic field. The prevailing relaxation mechanism for beads used in the present work is the Brownian relaxation mechanism. In the Brownian relaxation model,27 the characteristic relaxation time is given by τB ) 3ηVB /kT

(2)

where VB is the bead hydrodynamic volume, kT is the thermal energy, and η is the dynamic viscosity of the carrier liquid. The Brownian relaxation frequency, fB ) (2πτB)-1, is the frequency characterizing the position of the peak in the m′′ vs frequency spectrum. To account for relaxation time distributions, the Cole-Cole model28 gives the following empirical expression for the complex magnetization m(ω) ) (m0 - m∞)/(1 + (iωτB)1-R) + m∞

(3)

where R (the Cole-Cole parameter), 0 < R < 1, is a measure of the relaxation time distribution width; the narrower the distribution, the closer R is to zero. A more complete treatment of the theory is presented elsewhere.19,20,29,30 Quantitative Analysis Using the VAM-NDA Bioassay. Quantitative analysis of a single type of target-DNA can either be of the turn-off or turn-on type,20 where the former is based on the decrease of the HFP level while the latter refers to the increase of the LFP level with increasing RCA-coil concentration. With beads having a bare physical diameter of 40 nm, both turn-off and turn-on detection have been shown to be possible while only (25) Debye, P. Polar Molecules; The Chemical Catalogue Company: New York, 1929. (26) Ne´el, L. Ann. Geophys. 1949, 5, 99–136. (27) Brown, W. F., Jr. J. Appl. Phys. 1963, 34, 1319–1320. (28) Cole, K. S.; Cole, R. H. J. Chem. Phys. 1941, 9, 341–351. (29) Stro ¨mberg, M.; Gunnarsson, K.; Valizadeh, S.; Svedlindh, P.; Strømme, M. J. Appl. Phys. 2007, 101, 023911. (30) Stro ¨mberg, M.; Gunnarsson, K.; Johansson, H.; Nilsson, M.; Svedlindh, P.; Strømme, M. J. Phys., D: Appl. Phys. 2007, 40, 1320–1330.

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Figure 1. Illustrations of the single- and biplex target recognition, amplification, and detection reactions. (a) Padlock probes are mixed with a sample containing the target sequence (target 1). The probes are circularized guided by target 1, and the juxtaposed probe ends are joined in a highly specific ligation reaction. The DNA circles are amplified by RCA, which creates a single-stranded concatemer product that collapses into a micrometer-sized coil of DNA (RCA-coil). Magnetic beads functionalized with oligonucleotides complementary to the RCA-coils are added, and the beads are hybridized to the RCA-coils. This strongly affects the Brownian relaxation behavior of the beads. The peak response for free beads in the imaginary part of complex magnetization (m′′) vs frequency spectrum is referred to as the high-frequency peak (HFP), whereas beads incorporated in the RCA-coils respond by a low-frequency peak (LFP). The presence of a target is detected as a decrease in the proportion of free beads reflected as a decrease of the HFP amplitude (HFP level, turn-off detection) and sometimes an increase of the LFP amplitude (LFP level, turn-on detection). The diagram axes are m′′ (y-axis) and log(f) (x-axis) where f is the frequency of the applied ac magnetic field. (b) The absence of the correct target sequence does not give rise to DNA circles and RCA-coils, and all beads remain free; therefore, the HFP exhibits high amplitude in the diagram and the LFP is absent. (c) Biplex detection is enabled by applying a cocktail of padlock probes targeting different sequences. The RCA-coils are distinguished by the use of differently sized magnetic beads, which are equipped with target-specific oligonucleotides. The m′′ vs frequency curve corresponds to a composite curve that has been resolved into two HFPs, each belonging to one specific bead size, using a Cole-Cole model fit (deconvolution). The composite LFP, i.e., the total response from RCA-coils with hybridized beads, is neglected in the deconvolution model and therefore not illustrated in the diagram. (d) The absence of the correct target sequences does not give rise to DNA circles and RCA-coils, and all beads remain free. As in panel c, the composite curve has been resolved into its two HFP contributions using the Cole-Cole model.

turn-off detection applies for bead sizes of 130 and 250 nm.20 A single-target assay exhibits high quantification accuracy. To exemplify, for the single-target assay presented in ref 19 using 130 nm beads with data given in Figure 2 in this reference, the coefficient of variation (CV) values are 19%, 2.9%, 3.3%, 4.2%, and 2.7% for 3.7, 11, 33, 100, and 300 pM target concentrations, respectively. Assume now that k types of target-DNA are to be quantitatively detected simultaneously in the same compartment. After target recognition, ligation, and RCA, the sample may contain k types of RCA-coils for which k types of oligonucleotide detection probes can be designed where each probe matches, i.e., complementary to, exactly one type of RCA-coil. Each type of detection probe is 3400

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attached to one specific bead size, and the k types of detection probe functionalized beads are thereafter mixed together and added to the sample for probing the different concentrations of RCA-coils simultaneously. Upon this, each type of detection probetagged bead will hybridize to the RCA-coil it matches. As a consequence, the composite LFP will be the result of a cocktail of LFPs, including the individual contributions from the different k types of separate RCA-coils containing beads as well as contributions from coil clusters bound together by beads. It will be advantageous to choose the largest bead size such that its free bead relaxation peak occurs well above the composite LFP. With the use of RCA-coils of about 1 µm size, the former species have relaxation frequencies around 1 Hz whereas the latter species

Figure 2. m′′ vs frequency at 310 K for different concentrations of T250 (panel a, batch Ib used) and T130 (panel b, batch IIb used) visualizing the outcome of the T250 and T130 single-target assays. Sample preparation was incubation for 30 min at 343 K, and bead concentrations were the same as in the biplex assay. All samples include genomic background material. The lines are guides for the eye.

exhibit a relaxation frequency far below 1 Hz. For frequencies well above the composite LFP region, the composite m′′ vs frequency spectrum can to a good approximation to be expressed as a sum of k free bead Cole-Cole distributions, i.e., a sum of k HFPs m''composite(f) ) k

∑ j)1

(

(m0,j - m∞,j)(f/fB,j)1-Rj sin (1 - Rj) 2(1-Rj)

1 + (f/fB,j)

+ 2(f/fB,j)

1-Rj

(

π 2

)

cos (1 - Rj)

π 2

)

(4)

which can be deconvoluted to its k HFP contributions by fitting the experimental data to eq 4 by a nonlinear least-squares procedure. Thereby, the k HFP levels can be obtained and compared with the corresponding k negative control HFP levels. Figure 1 provides a schematic illustration of single-target detection (left) and biplex detection (right) using the VAM-NDA biosensor principle. EXPERIMENTAL SECTION Detection Probe Functionalization of Magnetic Beads and Characterization of Probe-Tagged Bead Batches. Three aqueous suspensions of relatively monodisperse amine-functionalized

cluster-type magnetic beads (nanomag-D NH2 ferrofluids) having physical mean diameters of 80, 130, and 250 nm were supplied from Micromod Partikeltechnologie GmbH. For the 80, 130, and 250 nm ferrofluids, the polydispersity indexes were 0.378, 0.067, and 0.115, respectively, as determined from photon correlation spectroscopy by the manufacturer. The 130 and 250 nm beads were used for quantitative biplex detection whereas all three sizes were used for a qualitative demonstration of triplex detection. Detection probe coupled single-sized bead batches were synthesized as described elsewhere;20 oligonucleotide surface coverages were determined by a fluorescencebased method,19 and detection batches were obtained by mixing single-sized batches in specific volume amounts. Measurements of the saturation magnetic moment at 310 K on 30 µL of each of batches I, II, and IV using a superconducting quantum interference device (SQUID) magnetometer (QD MPMS XL, Quantum Design) were performed in order to calculate the bead concentration (in units of picomolar) and the solid content of beads (in units of milligrams of beads per milliliter) in each batch. Properties of the batches are given in Table 1. For clarity, each target and detection probe, respectively, is abbreviated by “T” and “D” followed by the size of the bead used for the detection of the target, e.g., “T250” and “D250”, respectively. Padlock Probe Target Recognition, Ligation, and RCA. For biplex and triplex detection, padlock probe target recognition and ligation were performed separately for each bacterial DNAsequence target (target T250 recognized by padlock probe P250 and so forth) and the circularized complexes were thereafter mixed followed by RCA for 1 h, creating a mixture of either T250 and T130 coils (biplex detection) or of T250, T130, and T80 coils (triplex detection). Different amounts of the circularized complexes were used for obtaining samples with different RCA-coil concentrations. For T250 and T130 single-target assays, only one single type of circularized complex was RCA amplified, creating samples containing only RCA-coils corresponding to the T250 and T130 target, respectively. Detailed procedures of ligation and RCA are given in refs 19 and 20. Additionally, after RCA, genomic background material derived from blood belonging to a human male individual was added so that each magnetically characterized sample (see below) contained 150 ng of genomic background material. This was implemented in order to achieve similar sedimentation/adsorption conditions in all samples.20 Sequences are listed in Table 1 where T250, T130, and T80 denote the V. vulnificus, V. cholerae, and E. coli target sequences, respectively. Magnetic Probing of RCA-Coils. For probing the target concentrations in a biplex or triplex sample, 25 µL of the detection batch (batch III for biplex detection and batch V for triplex detection) was gently mixed at room temperature with 25 µL of solution containing RCA-coils or no coils in the case of a negative control sample, and 30 µL of the mixture was extracted for immediate characterization in the SQUID magnetometer. The characterization began with 30 min of incubation at 343 K followed by an ac magnetic moment (in units of electromagnetic unit, emu) measurement at 310 K in the frequency range 1000-0.5 Hz (ac excitation field amplitude 2 Oe). Finally, the saturation magnetic moment (in units of emu) was determined at the same temperature in order to calculate the magnetic bead content in the sample Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

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Used for the T250 assay. b Used for the T130 assay. c Used for the quantitative biplex assay (T250 and T130). d Used for the qualitative triplex assay (T250, T130, and T80). a

sequence

5′-TTGTAAAGCACTTTCAGTTGTGAGGAAGGT-3′ (Vibrio vulnificus sequence) 5′-ACTGAAAGTGCTTTACAACTTCTAGAGTGTACCGACCTCAGTAGCCGTGACTATCGACTCTGGACCTTAATCGTGTGCGACCTTCCTCACA-3′ SH-5′-TTTTTTTTTTTTTTTTTTTTCTGGACCTTAATCGTGTGCG-3′-FITC 5′-CCCTGGGCTCAACCTAGGAATCGCATTTG-3′ (Vibrio cholerae sequence) 5′-TAGGTTGAGCCCAGGGACTTCTAGAGTGTACCGACCTCAGTAGCCGTGACTATCGACTTGTTGATGTCATGTGTCGCACCAAATGCGATTCC-3′ SH-5′-TTTTTTTTTTTTTTTTTTTTGTTGATGTCATGTGTCGCAC-3′-FITC 5′-ACGTCGCAAGACCAAAGAGGGGGACCT-3′ (Escherichia coli sequence) 5′-CTTTGGTCTTGCGACGTCAGTGGATAGTGTCTTACACGATTTAGAGTGTACCGACCTCAGTAGCCGTGACTATCGACTAGGTCCCCCT-3′ SH-5′-TTTTTTTTTTTTTTTTTTTTGTGGATAGTGTCTTACACGA-3′-FITC

D80 80

name

D130 130

T250 padlock probe for T250 (P250) detection probe for T250 (D250) T130 padlock probe for T130 (P130) detection probe for T130 (D130) T80 padlock probe for T80 (P80) detection probe for T80 (D80)

3

14

186

average number of detection probes per bead solid content of beads (mg/mL)

672 8.26 10 µL of I and 110 µL of PBS mixed 2882 5.98 30 µL of II and 90 µL of PBS mixed 40 µL of I, 120 µL of II, and 320 µL of PBS mixed 3561 1.72 25 µL of I, 35 µL of II, 40 µL of IV, and 50 µL of PBS mixed

bead concentration (pM)

D250 250

I Iba II IIbb IIIc IV Vd

detection probe bare physical bead diameter (nm) batch

Table 1. Properties of Detection Probe Functionalized Bead Batches (Upper Half) and Sequences of Targets, Padlock Probes, and Detection Probes (Lower Half) 3402

(the diamagnetic contribution from the sample holder was subtracted). In the case of biplex detection, the corresponding single-target experiments, using batch Ib for T250 detection and batch IIb for T130 detection, were performed in the same manner. The corrected composite complex magnetization (in units of emu per gram of beads) profile for a biplex or triplex sample was calculated as mcomposite,corrected(f)[emu/g] ) mcomposite(f)[emu]

Ms,NC[emu] (5) Ms[emu]massbeads,NC[g]

where mcomposite(f) is the measured complex magnetic moment, Ms is the saturation magnetic moment for the composite sample, Ms,NC is the saturation magnetic moment for the negative control sample, and massbeads,NC is the total mass of beads in the negative control. In this expression, the factor Ms,NC/Ms appears in order to compensate for small variations in the amount of magnetic material from sample to sample and to ensure that each corrected composite complex magnetization spectrum corresponds to the same amount of magnetic material as in the negative control sample. In the case of biplex analysis, each corrected composite complex magnetization spectrum was deconvoluted as described below for simultaneous target quantification. RESULTS AND DISCUSSION Single-Target Assays. Figure 2 shows m′′ vs frequency spectra at 310 K for different concentrations of T250 (panel a, batch Ib used) and T130 (panel b, batch IIb used). m′ vs frequency spectra belonging to the data presented in Figure 2 can be found in the Supporting Information, Figure S1. These two single-target assays were designed to closely correspond to the T250 and T130 assays occurring in the same compartment (biplex detection), i.e., the same bead concentrations, sample preparation, and content of genomic background material were used. As concluded in ref 20, the presence of genomic background material impedes bead sedimentation and/or adsorption to the sample container walls. In Figure 2, for both the T250 and T130 cases, the HFP level decreases with increasing RCA-coil concentration whereas the LFP levels show no clear correlation with the RCA-coil concentration, i.e., the behavior is of turn-off type as observed in earlier work.19,20 Note that LFPs are visible in the T130 case (at about 1 Hz), most pronounced for 100 pM target concentration, a fact which will be discussed further below in connection with the deconvolution procedure of the composite m′′ vs frequency biplex spectra. Fits according to the Cole-Cole model, see eq 3, were performed on the HFPs in Figure 2, and detailed results can be found in the Supporting Information, Table S1 and Figure S2. Figure 3 displays the HFP Brownian relaxation frequency (a), the HFP Cole-Cole parameter (b), and the HFP level (c) vs target concentration for the single-target assays. The inset in Figure 3c shows HFP levels vs target concentration normalized with respect to the negative control HFP level from which it follows that the two kinds of probe-tagged beads have similar immobilization efficiencies with that of the 250 nm beads being somewhat higher for higher RCA-coil concentrations. In Figure 3a, it can be observed that the HFP Brownian relaxation frequency increases slightly with increasing RCA-coil concentration for the T250

Figure 4. Corrected composite m′′ vs frequency curves at 310 K visualizing the outcome of the T250, T130 biplex experiment (batch III used, sample preparation was incubation for 30 min at 343 K). The lines are guides for the eye.

Figure 3. Panels a, b, and c show Brownian relaxation frequencies, Cole-Cole parameters (R), and HFP levels, respectively, for the HFPs in Figure 2. The first two parameters were obtained from Cole-Cole fits whereas the third represents the maximum values of the HFPs. Linear fits to the data are displayed in panels a and b. The inset in panel c shows HFP levels normalized with respect to the negative controls.

experiment (250 nm beads) whereas it decreases slightly in the T130 experiment (130 nm beads) and the trends are well described by linear expressions. These observations suggest that in the case of 250 nm mean bead size (T250 experiment), there is preferential immobilization of larger beads in the size distribution and/or bead aggregates whereas the opposite holds for the case with 130 nm beads (T130 experiment). These observations

are consistent with results (unpublished) obtained from experiments where the size distribution of the nonimmobilized bead population for a number of different RCA-coil concentrations was analyzed by dynamic light scattering. Detailed investigations of these phenomena are however beyond the scope of this paper and require further studies. Using beads with a narrower size distribution would most likely give smaller variations in the HFP Brownian relaxation frequency values when varying the RCA-coil concentration. As can be seen in Figure 3b, the HFP Cole-Cole parameter increases slightly with increasing RCA-coil concentration in both the T250 and T130 cases and also these trends are well described by linear expressions. In other words, a higher RCA-coil concentration gives a broader distribution of relaxation times for the remaining nonimmobilized bead population. A reasonable explanation for this is that positively charged (protonated) amine groups on the surface of the nonimmobilized beads (pH ∼ 7) interact electrostatically with the negatively charged RCA-coil DNA chains. This implies that a bead interacting with a coil electrostatically, without immobilization via base-pair hybridization, exhibits a slightly different Brownian relaxation behavior compared to a completely free bead, something which gives rise to a slight HFP broadening. For clarity, because of the particular oligonucleotide coupling chemistry used, the amine groups are situated either on iodoacetamide alkylated surface sites or appear as nonreacted functionalities on the dextran surface of the beads (see ref 30). It should also be mentioned that a detection probe on a bead in close vicinity of an RCA-coil has to be in a very specific position with respect to the RCA-coil DNA chain in order to hybridize. This implies that a probe-tagged bead in close vicinity of a coil is able to interact electrostatically with the coil without necessarily immobilizing. Biplex Detection. Figure 4 shows corrected composite m′′ vs frequency spectra at 310 K visualizing the outcome of the quantitative T250, T130 biplex experiment (batch III used). m′ vs frequency spectra belonging to the data presented in Figure 4 are shown in the Supporting Information, Figure S3. A total of 12 different T250, T130 concentration combinations were evaluated. In Figure S3 in the Supporting Information, it can be observed that the magnitude of m′ tends to decrease when the sum of the target concentrations increases, which is reasonable since this quantity correlates with the total amount of immobilized beads.19 In this biplex experiment, the two HFPs, one corresponding to Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

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free 250 nm beads (T250 detection) and the other to free 130 nm beads (T130 detection), are separated only by one decade of frequency implying a rather large HFP overlap. For the case k ) 2 in eq 4, we have in total six fitting parameters, i.e., m0,T250 - m∞,T250, fB,T250, RT250, m0,T130 - m∞,T130, fB,T130, and RT130. To improve on accuracy in the fitting procedure, i.e., the deconvolution of the composite biplex spectra in Figure 4, some parameters should be locked at predetermined values. First, it is reasonable to assume that fB,T250 and fB,T130 are close to the Brownian relaxation frequencies determined in the corresponding single-target experiments performed separately, see Figure 3a, since these parameters are related to the mean hydrodynamic size of the nonimmobilized magnetic beads. It should be mentioned that it was carefully checked that there was no cross-hybridization between the T250 and T130 detection probes and the T250 and T130 RCA-coils. This was established in experiments excluding magnetic beads by attaching RCA-coils (∼1 µm size), derived from T250 sequence/ T250 padlock probe (T130 sequence/T130 padlock probe) circularized complexes, on glass-substrates followed by hybridization by T130 (T250) detection probes while studying the surfaces using fluorescence microscopy. Here, hybridization reactions between coils and detection probes would have given rise to micrometer sized bright spots in the fluorescence microscope, i.e., confined clusters of fluorophores (see, e.g., ref 31). Second, from similar arguments as above in connection to the single-target experiments, it is reasonable to assume that the presence of T250 RCA-coils may affect the broadening of the T130 HFP and vice versa, i.e., RT250 and RT130 should be free fitting parameters. Thus, the curve fitting was performed using the Brownian relaxation frequency values from the linear fitting curves in Figure 3a and keeping the R and the m0 - m∞ parameters free. Raw data is provided in the Supporting Information, Table S2 and Figure S4. Figure S5 in the Supporting Information shows HFP levels (a, b) and Cole-Cole parameters (c, d) vs target concentration obtained from the fitting procedure of the composite biplex spectra in Figure 4, indicated by b. In each panel, the concentration of the opposite target is displayed at each data point. × represent HFP levels from the single-target assays (Figure 3c), which were recalculated to account for the fact that the HFP levels in Figure S5 in the Supporting Information are given in units of emu per gram of mixture of the two bead sizes whereas the HFP levels in Figure 3 are given in units of emu per gram of one single bead size. Figure 5 displays average T250 (panel a) and T130 (panel b) HFP levels vs target concentration obtained from the deconvolution of the composite biplex spectra displayed in Figure 4. The indicated error bars, obtained by varying the concentration of the opposite target, represent absolute deviations equal to difference between the largest and smallest HFP level value obtained for each target concentration; see Supporting Information, Figure S5, panels a and b, for detailed data. As can be observed in Figure S5, panels a and b in the Supporting Information, the single-target negative control HFP levels are somewhat lower in magnitude than the biplex negative control levels, an observation which most likely can be explained by a lower sedimentation rate and/or bead adsorption tendency in the (31) Jarvius, J.; Melin, J.; Go ¨ransson, J.; Stenberg, J.; Fredriksson, S.; GonzalezRey, C.; Bertilsson, S.; Nilsson, M. Nat. Methods 2006, 3, 725–727.

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Figure 5. Average T250 (panel a) and T130 (panel b) HFP levels vs target concentration obtained from deconvolution of the composite biplex spectra displayed in Figure 4. The error bars represent absolute deviations indicating the difference between the largest and smallest HFP level value obtained for each target concentration by varying the concentration of the opposite target. A detailed description of the data points lying behind the calculation of each error bar can be found in the Supporting Information, Figure S5, panels a and b. Data points lacking error bars represent single measurements for which the concentration of the opposite target has not been varied.

biplex negative control sample. In ref 20, it was found that genomic background material impedes bead sedimentation and/or bead adsorption and analogously, in the biplex negative control sample, the second type of beads may act as background and thus impede the sedimentation and/or adsorption of the first type of beads and vice versa. Furthermore, the single-target HFP levels tend to be somewhat lower in magnitude than the corresponding biplex levels and the spread in HFP levels is larger in the T130 case (Figure S5b in the Supporting Information). Moreover, in the T130 case, at a given T130 concentration, the HFP levels tend to be higher for a higher T250 concentration whereas the T250 HFP levels (Figure S5a in the Supporting Information) are somewhat more insensitive to T130 concentration variations. These observa-

tions are possibly related to the bead/coil attractive electrostatic interaction discussed in connection to the single-target assays above. More precisely, since probe-tagged beads may interact electrostatically with matching RCA-coils as well as with nonmatching RCA-coils, the presence of nonmatching coils may affect the hybridization between the probe-tagged beads and their matching RCA-coils. This interpretation is supported by the fact that the 130 nm beads are affected more by the presence of nonmatching coils than the 250 nm beads, where the latter kind of beads have a considerably larger amount of oligonucleotides per surface area unit, see Table 1, where the oligonucleotides “screen” the positively charged amine groups. From the Supporting Information, Figure S4, for the “T250 300 pM, T130 300 pM” sample, it is obvious that the curve fit is rather poor, most likely because the composite LFP contributes significantly to the composite m′′ vs frequency spectrum, implying that three relaxation peaks have to be taken into account in order to obtain a reliable deconvolution. Therefore, 300 pM data points, especially for 300 pM T130 concentration, should be considered with caution. As can be seen in Figure S5, panels c and d in the Supporting Information, for a given concentration of one target, the Cole-Cole parameter increases slightly with increasing concentration of the opposite target. The interpretation of this is similar to the one of the HFP broadening effects in the single-target experiments. In summary, the T250 HFP levels (Figure 5a), are found to be rather insensitive to variations of T130 target concentration in the biplex detection experiment. Roughly, a doubling of the T250 target concentration in the concentration range between 10 and 100 pM can be assessed. As well, since the spread in T130 HFP levels exhibits a systematic pattern (see Figure S5b in the Supporting Information), a T130 HFP level adjustment procedure may be implemented after deconvolution to improve the T130 quantification accuracy. In this, the obtained T130 HFP level should be adjusted downward if the T250 concentration is found to be 100 pM or higher. Additionally, with the use of beads with a more narrow size distribution, the spread in HFP levels may be further decreased. A more accurate deconvolution could probably be achieved by increasing the separation of the HFPs. This could in turn be accomplished by using beads of as large size difference as possible, which would require a magnetometer with a larger frequency window. Furthermore, a higher coverage of detection probes should be used in order to screen unwanted electrostatic interactions between beads and nonmatching coils. Also, a buffer of a higher pH would decrease the strength of the electrostatic bead/coil interactions. For clarity, it should be mentioned that the observed spread in T250 and T130 HFP levels in Figure S5, panels a and b in the Supporting Information, mainly appear as a consequence of that the presence of the other kind of RCA-coil interferes with the detection of the first kind of RCA-coil and vice versa. Thus, biplex detection performed with the particular beads used in this study needs to be optimized further to give as good quantitative results as obtained in a single target assay,19 but the results presented above show that the procedure in principle can be used to quantify biomolecules in multiplex assays. However, in its current format, biplex detection is able to give qualitative information. This becomes obvious by comparing the m′′ vs frequency spectra for the T250 0 pM, T130 0 pM (negative control) and T250 300 pM, T130 300 pM samples in Figure 4.

Figure 6. Corrected composite m′′ vs frequency curves at 310 K visualizing the outcome of the T250, T130, T80 triplex experiment (batch V used, sample preparation was incubation for 30 min at 343 K). The lines are guides for the eye.

Brief Qualitative Demonstration of Triplex Detection. Figure 6 shows the corrected composite m′′ vs frequency spectra at 310 K visualizing the outcome of a brief qualitative demonstration of T250, T130, T80 triplex detection (batch V used). The corresponding corrected composite m′ vs frequency spectra are given in the Supporting Information, Figure S6. Five different T250, T130, T80 concentration combinations were evaluated. The detection batch was prepared by choosing the relative amount of the different beads so that in the “T250 0 pM, T130 0 pM, T80 0 pM” sample (negative control), the T250 HFP is roughly twice as high as the T130 and T80 HFPs. This resulted in a single broad plateaulike composite m′′ vs frequency spectrum for the negative control. In Figure S6 in the Supporting Information, as expected, it can be observed that the magnitude of m′ decreases when the sum of the target concentrations increases. Also, the “T250 0 pM, T130 0 pM, T80 300 pM” m′ curve is situated above the “T250 0 pM, T130 300 pM, T80 0 pM” curve which mainly can be explained by the fact that the 80 nm beads have a lower immobilization efficiency than the 130 nm beads due to a considerably lower detection probe surface coverage (Table 1), consistent with earlier observations.20 In Figure 6, it can be seen that the “T250 300 pM, T130 300 pM, T80 300 pM” sample exhibits a much suppressed and weakly frequency dependent m′′ vs frequency spectrum where the T250 and T130 HFPs almost have disappeared and only a small T80 HFP is visible around 130 Hz. For the “T250 300 pM, T130 0 pM, T80 300 pM” sample, the T250 and T80 HFPs have strongly diminished in height compared to the negative control and a peak is visible around 100 Hz, mainly corresponding to free 130 nm beads but a small contribution from nonimmobilized 80 nm beads cannot be ruled out. For the “T250 0 pM, T130 300 pM, T80 0 pM” sample, the T130 HFP has strongly diminished in height compared to the negative control and the composite m′′ vs frequency spectrum is essentially a superposition of two HFPs, each corresponding to free 250 and 80 nm beads, respectively. The interpretation of the composite m′′ vs frequency spectrum for the “T250 0 pM, T130 0 pM, T80 300 pM” sample is similar. From similar arguments as presented above for biplex detection, triplex detection performs well qualitatively. SUMMARY AND CONCLUDING REMARKS The possibility to perform quantitative biplex detection of V. vulnificus and V. cholerae DNA-sequences using 250 and 130 nm magnetic beads by using the VAM-NDA bioassay was investigated. Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

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In summary, k-plex detection using the VAM-NDA method involves the simultaneous detection of k types of RCA-coils in one compartment. This is accomplished by choosing k types of oligonucleotide detection probes and k different sizes of magnetic beads exhibiting Brownian relaxation behavior. Each type of detection probe is attached to one specific bead size, and the k types of detection probe functionalized beads are mixed together. We have demonstrated the detailed methodology for biplex detection which includes the use of a detection batch consisting of two bead sizes and a nonlinear least-squares deconvolution method of the composite m′′ vs frequency spectra based on the Cole-Cole model. The results have been compared with the results obtained from the corresponding single-target assays performed separately. We have also given a brief qualitative demonstration of triplex detection of V. vulnificus, V. cholerae, and E. coli DNA-sequences using a detection batch consisting of a mixture of detection probe functionalized 250, 130, and 80 nm beads. Contrary to single-target assays which exhibit very high quantification accuracy, we found that the biplex assay gives a rather large spread in HFP levels when keeping one target concentration constant while varying the other, possibly due to attractive electrostatic interactions between positively charged amine groups on the bead surface and negatively charged DNA in nonmatching RCA coils. In this study, for biplex and triplex detection, padlock probe target recognition and ligation were performed separately for each target and the circularized padlock probes were thereafter mixed followed by RCA. By this, samples consisting of mixtures of several types of RCA-coils were obtained. Alternatively, for simultaneous selective analysis of several sequence species, a mixture of padlock probes designed to detect different targets can be applied to the ligation reaction.31 The quantification accuracy may be improved by using beads with a more narrow size distribution and higher detection probe coverage on bead surfaces in order to screen the above-mentioned electrostatic interactions. A more alkaline buffer environment may be used in order to decrease the number of protonated amine groups on the beads, thereby decreasing the strength of the attractive electrostatic bead/coil interaction. Additionally, choosing beads that differ more in size in order to obtain HFPs more

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separated in frequency would give a more accurate deconvolution. For this a magnetometer with a larger frequency window is required. Conclusively, biplex detection performed with the VAMNDA method needs to be optimized further to give as good quantitative results as obtained in single target assays, but the results presented here show that the procedure is promising and may become a valuable tool for, e.g., multiplex detection of infectious pathogens and for multiplex quantification of biomolecules detected with probing techniques that generate DNA circles, permitting inclusion of an internal control for improved quantification accuracy. Also, the biplex and triplex assays perform well qualitatively in their current formats, i.e., they are able to provide yes/no answers concerning the presence of targets. Finally, it should be mentioned that the SQUID magnetometer used in this paper has to be replaced by a low-cost miniaturized sensor device able to measure the complex magnetization in order to achieve a commercial biosensor device. The complete device should also include units such as microfluidic channels and compartments for reagent storage. Nevertheless, we have provided a proof-of-principle of multiplex detection using the VAMNDA biosensor principle which is a major step in the biosensor development. ACKNOWLEDGMENT The Knut and Alice Wallenberg Foundation (KAW), the Swedish Foundation for Strategic Research (SSF), the Swedish Defense Nanotechnology Programme, and the Swedish Research Council (VR) are gratefully acknowledged for their financial support. SUPPORTING INFORMATION AVAILABLE Detailed curve fit results for the T250 and T130 single-target experiments and for the T130, T250 biplex experiment; m′ vs frequency spectra belonging to the data presented in Figures 2, 4, and 6. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 17, 2008. Accepted March 20, 2009. AC900561R