Magnetic nanobeads present during enzymatic amplification and labeling for a simplified DNA detection protocol based on AC susceptometry

Magnetic biosensors are promising candidates for low-cost point-of-care biodiagnostic devices. For optimal efficiency it is crucial to minimize the time and complexity of the assay protocol includi ...


I. INTRODUCTION
In recent years, much research has focused on nanomaterials and their ability to elevate the performance of various biosensor techniques used in diagnostic applications. 13][4] Apart from requirements concerning sensitivity, selectivity and reliability, biosensors should ideally also be easy to handle and provide a fast response. 5,6Furthermore, integration and automation of all reaction steps in sample preparation with sample analysis is essential for successful development of a fully automated diagnostic test.The possibility to implement such a test is much dependent on the simplicity of the assay.For instance, a homogeneous assay including as few steps and/or changes in variables as possible is desirable and integration of complex multistep protocols has been challenging to date. 7Hence, magnetic micro-and nanoparticles have been proven useful for automation of sample preparation in bioanalytical devices by serving as solid phase supports to enable reagent exchange and washing in multi-step assays. 8,9agnetic biosensors, usually classified as either surface or volume based, are potential candidates for low-cost biodiagnostic devices operating in outpatient and point-of-care settings including applications such as infectious diagnostics, 10 food safety 11 and drinking water quality assessment. 12urface-based methods typically utilize thin film magnetic sensor elements functionalized with biomolecular probes and are therefore only sensitive to magnetic nanoparticles in the close vicinity to the sensor surface. 13In contrast, volume-based sensors probe the entire sample volume and offer a convenient homogeneous readout format.One type of volume-based magnetic biosensing relies on measuring the decrease in Brownian relaxation frequency upon probe-target binding (e.g. an antigen-antibody reaction) occurring on the surface of magnetic nanobeads. 14,15n 2008 we demonstrated a refined and improved version of this approach for detection of DNA target sequences. 16The assay protocol begins with highly specific target recognition through padlock probe ligation forming DNA circles.Thereafter, the circles serve as templates for isothermal rolling circle amplification (RCA, typically for 60 min at 37 • C) generating macromolecular single stranded DNA coils (size about 1 µm) with a repeating sequence that is complementary to that of the padlock probe.Subsequently, the DNA coils are post-labeled in a separate reaction step in high salt buffer at elevated temperature (typically for 20 min at 55 • C) with magnetic nanobeads (size around 100 nm) conjugated with single-stranded DNA probes complementary to the repeating motif of the DNA coils.Binding of beads to DNA coils gives rise to a suppression of the AC magnetic susceptibility χ = χ ′ − i χ ′′ , where χ and χ are the in-phase and out-of-phase components of the AC susceptibility, respectively.More specifically, the binding of beads give rise to a decrease in amplitude of χ at the Brownian relaxation frequency of non-bound beads, χ ′′ max .Non-complementarity between the probes on the beads and the DNA coil sequence does not give rise to any measurable attractive binding interaction i.e. the AC magnetic susceptibility response is equal to that of a blank control sample (absence of DNA coils). 168][19] We are currently focusing on developing a device, including automated sample preparation, for infectious diagnostics based on the bioassay principle using either a miniaturized AC susceptometer or a chip-based high-temperature superconducting quantum interference device.In connection to this work it is highly relevant to strive towards automation of the molecular protocol by investigating the possibilities for protocol simplifications.
Herein, the possibility of labeling DNA coils in real-time, i.e. adding magnetic nanobeads to the RCA mix with the intention to obtain bead-coil hybridization already during the RCA reaction, is evaluated.This approach, hereafter denoted as a combined protocol, aims at reducing the number of protocol steps as well as assay time and possibly also improving the limit of detection (LOD) of the assay.Also, a novel approach to analyze AC susceptibility data by recording the phase angle, ξ = arctan ( χ ′′ / χ ′ ), is investigated, avoiding the need of normalization of AC susceptibility data.

II. MATERIALS AND METHODS
Sequences of targets, padlock probes and detection oligonucleotides can be found in Table I.Note that concentrations of reagents given below, unless otherwise stated, refer to stock concentrations.A. Detection probe conjugation of magnetic nanobeads 40 µL of magnetic nanobead solution (BNF-Starch streptavidin, 100 nm, Micromod, Germany) of 10 mg/mL was washed two times with a special washing buffer (final composition of 10 mM Tris-HCl [Sigma-Aldrich, USA], 5 mM EDTA [Sigma-Aldrich], 0.1% Tween20 [Sigma-Aldrich], and 0.1 M NaCl [Sigma-Aldrich]).2.49 µL of 10 µM detection probe solution (Biomers, Germany, 60 fold excess of probes with respect to the total number of beads) was added followed by incubation at room temperature during 15 min.Finally the bead suspension was washed two times with 1×phi29 buffer (Thermo Fisher Scientific, USA) and resuspended in 40 µL of 1×phi29 buffer (∼10 mg/mL of beads).A shorter (probe 1, melting temperature T m =54 • C) and a longer (probe 2, T m =75 • C) detection probe hybridizing to the DNA coils by 22 and 35 bases, respectively, were examined in this study, see Table I.
To determine the LOD for the optimized combined protocol (detection probe 1, 100 mM final concentration of NaCl in the RCA mix) and for post-labeling (detection probe 1), an RCA master mix was prepared by mixing 150 µL phi29-buffer (10×), 90 µL dNTP (2.5 mM), 150 µL BSA (2 µg/µL), 150 µL NaCl (1M) and 855 µL MQ water.For the combined protocol 46.5 µL master mix, 0.5 µL ligated circles (20, 8, 2 nM) or water (in case of negative control), 1 µL phi29 polymerase (10 U/µL) and 2 µL of beads were mixed followed by incubation for 60 min at 37 • C and an end step of 5 min at 55 • C. 150 uL PBS was added prior to magnetic read-out.For post-labeling 46.5 µL master mix, 0.5 µL ligated circles (20, 8, 2 nM) or water (in case of negative control) and 1 µL phi29 polymerase (10 U/µL) were mixed followed by incubation for 60 min at 37 • C and an end step of 5 min at 65 • C. Thereafter 2 µL of beads were added followed by incubation for 20 min at 55 • C and addition of 150 µL PBS prior to magnetic read-out.

C. AC susceptometric read-out and data analysis
The frequency-dependent magnetic susceptibility, χ = χ ′ − i χ ′′ , was measured from 10 Hz to 100 kHz at room temperature in a commercial AC susceptometer system, DynoMag (Acreo, Göteborg).Target quantification (dose-response curves) was accomplished in two ways; either by considering (i) the decrease of χ ′′ max / χ ′ high upon increasing DNA coil concentration where χ ′ high is the frequency independent high frequency limit of χ ′ (proportional to the iron-oxide content in the sample) or (ii) the decrease of the phase angle ξ at high frequencies upon increased DNA coil concentration.

III. RESULTS AND DISCUSSION
Figure 1 summarizes the results from the optimization of the combined protocol where the relative peak decrease of a 50 pM DNA coil sample compared to the negative control, i.e. [  , is plotted versus different protocol parameters.A special notation is used to specify employed protocol parameters, t 1 @T 1 +t 2 @T 2 , where t 1 and t 2 denote the times (in minutes) for the RCA and the end step, respectively, and T 1 and T 2 are the corresponding temperatures (in • C).Panel (a) displays the relative peak decrease vs. NaCl concentration in the RCA mix where beads were conjugated with probe 1.Panel (b) shows the relative peak decrease vs. T 1 where the NaCl concentration in the RCA mix was 100 mM, t 1 =60 min, with or without a 5@55 end step and beads were conjugated with probe 2. Post-labeling reference points are also included.First of all, by comparing the relative peak decrease for post-labeling and 60@37+20@55 for 100 mM NaCl in panel (a), it can be concluded that the magnetic beads do not significantly interfere with the RCA reaction.Possible interference mechanisms could for instance be leakage of different kinds of metallic ions from the beads with an inhibitory effect on the phi29 enzyme or adsorption of enzyme on the bead surface.The lack of significant interference means that the starch casing of the beads is sufficient for the protocol.Furthermore, a comparison of the FIG. 1. Optimization of a biodetection protocol relying on the hybridization of detection probe-conjugated magnetic nanobeads (size 100 nm) to rolling circle amplification (RCA) products (DNA coils) with nanobeads present in the RCA reaction, i.e. a combined protocol.The combined protocol comprises the steps of running the RCA reaction for t 1 (min) at a temperature T 1 ( • C), followed by a t 2 min end step during at an elevated temperature T 2 ( • C).A notation "t 1 @T 1 +t 2 @T 2 " is used for these particular settings.Panel (a) displays the relative peak decrease (50 pM DNA coil sample compared to the negative control) vs. NaCl concentration in the RCA mix (beads conjugated with detection probe 1).Panel (b) shows the relative peak decrease vs. T 1 where the NaCl concentration in the RCA mix was 100 mM, t 1 =60 min, with or without a 5@55 end step (beads were conjugated with detection probe 2).Post-labeling refers to hybridizing the nanobeads to the DNA coils after completion of the RCA by employing 20 min of incubation at 55 • C. Measurements were repeated once (qualitative interpretation).60@37+5@65 points in panel (a) indicates that a NaCl concentration of about 100 mM is optimal for the RCA reaction.The existence of an optimum is the result of a compromise between two competing factors; viz. 1) the decrease in number and average length of DNA coils due to suppression of the enzymatic activity of the polymerase in the presence of NaCl and 2) the increase in bead-coil hybridization efficiency upon increasing NaCl concentration.It can also be concluded from panel (a) (by comparing the different 100 mM NaCl samples) that an insignificant number of beads hybridize to the DNA coils while the RCA reaction is ongoing and, consequently, that the major part of the bead-coil binding occurs during the end step.It is further found that an end step temperature of 55 • C, which is close to the melting temperature of detection probe 1, is more appropriate to use than the higher temperature of 65 • C; as the relative peak decrease for 60@37+10@55 and 60@37+5@55 is larger than for 60@37+10@65 and 60@37+5@65.There is also a trend of a decreasing amount of hybridized beads upon decreasing RCA time.For T 2 = 55 • C it can be concluded that t 1 = 5 min is enough to reach a steady state in the bead-coil binding reaction (the relative peak decrease for 60@37+5@55 and 60@37+10@55 are almost equal) and the amount of bound beads is nearly identical to that of post-labeling.In order to further explore the possibility to achieve bead-coil binding during the RCA process, a longer detection probe was evaluated, see panel (b).It was hypothesized that a detection probe with a higher T m would be able to hybridize more efficiently to the RCA product at 37 • C during amplification.Clearly, however, this is not the case and including an end step is also found to be necessary in this case.The most likely explanation for this phenomenon is that the rate of the amplification reaction and the coiling up of the single stranded RCA product is higher than that of the hybridization reaction between the detection probes and the RCA products, thereby not substantially increasing the number of available probe binding sites.Alternatively, there is a possibility that bead-coil hybridization occurs at a very early stage of the RCA process and that these bound beads sterically inhibit binding of additional beads while the DNA coils continue growing.DNA coils growing around bound beads could cause such steric hindrance since the beads hybridize to the DNA coils in a multivalent manner.A slight increase of T 1 from 37 to 45 • C does not improve the performance of the assay when an end step is used, while a significant improvement is obtained in the no-end-step case.Introducing gentle shaking of the RCA mix during the RCA reaction in the no-end-step experiments did not improve the bead-coil binding efficiency.Furthermore, T 1 = 50 • C is obviously too high temperature for the phi29 enzyme.Conclusively, it was found that a combined protocol with 60@37+5@55 conditions is optimal to use with nanobeads equipped with probe 1.

Reuse of AIP
Figure 2 presents a comparison between the two methods used for obtaining dose-response curves from frequency-dependent magnetic susceptibility spectra measured on samples prepared by postlabeling (as described in the last paragraph of section II, part B).Panels (a) and (b) show normalized χ ′′ spectra and phase angle spectra, respectively, for different DNA coil concentrations.Panel (c) displays dose-response curves, viz.χ ′′ max / χ ′ high (left y-axis) and phase angle taken at 2069 Hz (right y-axis) vs.DNA coil concentration.Apparently, the two methods give the same LOD (20 pM) but phase angle analysis constitutes a more convenient approach since the normalization of χ ′′ max with respect to χ ′ high can be avoided.Target quantification based on the phase angle method could also be performed by measuring the susceptibility at one single frequency.Thereby the read-out time in the DynoMag system would be substantially reduced and limited to about 2 min.
Based on the observation that the major part of the bead-coil hybridization in the combined protocol occurs during the end step, the LOD is expected to be comparable with post-labeling.This statement is confirmed by the results presented in Fig. 3 showing phase angle versus frequency spectra for the optimized combined protocol (a) and for post-labeling (b).It can be seen that the LOD equals 20 pM for both protocols.Compared to the previously used post-labeling protocol the combined protocol is faster and easier to automate.

IV. CONCLUSION
In this paper, possibilities for protocol simplifications for a biodetection principle relying on hybridization of probe-functionalized magnetic nanobeads to rolling circle amplification products have been evaluated.It can be concluded that beads can be present during amplification without noticeably interfering with the phi29 enzyme and that bead-coil hybridization subsequently is achieved during an end step at elevated temperature within just a few minutes prior to read-out in an AC susceptometer setup.Therefore one reaction step requiring an additional reagent handling step can be avoided, facilitating assay integration into automated formats.The reason for why the major part of bead-coil hybridization occurs during the end step and not during the RCA reaction is most likely related to that the rate of the RCA reaction and coiling up of the single stranded RCA product is higher than that of the hybridization reaction between the detection probes and the RCA products.Moreover, by recording the phase angle at a single frequency the total assay time for the optimized combined protocol (including padlock probe ligation, amplification, labeling and read-out) would be no more than 1.5 hours which is clinically relevant for a number of diagnostic applications, • C and an end step of 5 min at 65 • C. 20 µL of a hybridization buffer solution (2 µL Tris-HCl pH 8.0 [1 M], 4 µL EDTA [0.5 M], 1 µL Tween-20 [10%], 10 µL NaCl [5 M] and 3 µL MQ water) was finally added and the 4 nM DNA coil solution was diluted stepwise with hybridization buffer to lower concentrations (8 nM and 2 nM).Post-labeling was done by adding 2 µL of beads to 50 µL of DNA coil solution followed by 20 min incubation at 55 • C and addition of 150 µL PBS prior to magnetic read-out.

FIG. 3 .
FIG. 2.Comparison of two methods used for obtaining dose-response curves from frequency-dependent magnetic susceptibility data (χ = χ ′ − i χ ′′ ).The data correspond to post-labeling of DNA coils, using nanobeads with detection probe 1, a 60 min at 37 • C RCA step followed by addition of beads and incubation for 20 min at 55 • C prior to magnetic read-out.(a) χ ′′ spectra for different DNA coil concentrations normalized with respect to χ ′ high , the frequency independent high frequency limit of χ ′ .(b) Phase angle, ξ, spectra for different DNA coil concentrations where ξ = arctan(χ ′′ /χ ′ ).(c) Dose-response curves, viz.χ ′′ max /χ ′ high (left y-axis) and ξ taken at 2069 Hz (right y-axis) vs.DNA coil concentration.Error bars represent one standard deviation based on three independent measurements.

TABLE I .
Oligonucleotide sequences.Segments of the detection probes hybridizing to the DNA coils are highlighted in red.