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2 Typing of Viral DNA with Single Nucleotide Resolution

2 Typing of Viral DNA with Single Nucleotide Resolution

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A.I. Gomez and I.V. Smolina



Potential intertypic recombinants of both EBV types are present within the BC-1

cell line (Yao et al. 1998; Aguirre and Robertson 1999, 2000; Horenstein et al.

1997), so the LMP-1 probe (common for both types of EBV) and the EBNA-3(G)

probe (specific for EBV type 1) were applied to determine type specificity of the

EBV. The zero mismatch tolerance of PNA-based detection has previously been

demonstrated (Demidov et al. 2001; Smolina et al. 2010); the SNP in the EBV type

2 EBNA-3 gene is enough to prevent probe ligation and subsequent

RCA. Fluorescence from the EBNA-3(G) detection was observed in approximately

half of the BC-1 cells while detection of LMP-1 (common to both EBV types 1 and

2) produced a fluorescent signal in all cells (data not shown). The negative control

CCRL-CEM (EBV-) cells gave no fluorescent signal.



4



Quantitation of Gene Copy Number in Microfluidic

Droplet Format



In order to identify chromosomal variations such as copy number variations (CNVs)

and SNPs among a population of cells, analysis at the single-cell level is required

(Zong et al. 2012). High-throughput and multiparameter approaches are extremely

important for detecting cell-to-cell variability and exploring the interdependence of

cellular processes within individual cells of a heterogeneous population

(Haselgrubler et al. 2014). Microfluidic droplet systems provide a versatile platform

for biomedical research applications because they serve as high-density microreactors with pico or nanoliter volumes of samples and reagents (Agresti et al. 2010;

Kiss et al. 2008; Koster et al. 2008; Konry et al. 2011a, b). Using this technology,

cells and specific reagents can be encapsulated within single droplets where mixing

and reaction rates are enhanced and contamination is eliminated. The droplet-based

microfluidic system can be combined with PNA-assisted RCA to detect single-base

changes in human genomic DNA within a cells-in-flow format.

Genetic variations such as CNVs and aneuploidy are well-known indicators of

biological dysfunction and deregulation of molecular pathways in numerous malignancies. For example, increased copy number of mtDNA has been observed in

blood samples from breast and non-small cell lung cancer patients (Hosgood et al.

2010; Shen et al. 2010). Elevated copy numbers are also present in tumor tissues

from ovarian, endometrial, and head/neck cancer (Wang et al. 2005, 2006; Jiang

et al. 2005). Aneuploidy, on the other hand, has been linked to resistance of anticancer treatments and poor outcome in breast cancer patients (Ben-Porath et al. 2008).

Therefore, cytometric determination of DNA content within individual cells may

play an important role in medical diagnosis. Genomic DNA targeting via PNAassisted RCA can be applied in a droplet-based microfluidic system for fast and

accurate determination of DNA content in individual cells.

To implement the detection scheme in a one-step, droplet-based microfluidic

system the reagents for PNA-assisted RCA were incorporated within monodisperse

aqueous-emulsion nanoliter droplets containing single cells. Detection of singlecopy LMP1 and EBNA-3 and multiple copy EBNA-2IR target sites was performed



10



PNA-Assisted Rolling Circle Amplification for Detection of DNA Marker…



117



to test for quantitative capability of the approach. For single-cell resolution, PNA

invasion was performed and the cells were mixed with the oligonucleotide probes

and encapsulated in distinct nanoliter droplets of RCA reagents. The PDMS microfluidic system was used for single-cell encapsulation; it contained the droplet generation chip and an in-channel incubation chamber (Agresti et al. 2010; Koster et al.

2008; Konry et al. 2011a). The RCA reaction was performed at 37 °C for 2 h within

individual droplets in the incubation channel. The intensity of the fluorescent signal

was used to determine the number of target sites per genome of each cell.

Figure 10.3c, d shows the phase-contrast and fluorescent images of the droplets for

both the EBV-positive BC-1 cells and EBV-negative CCRL-CEM cells.

Quantification of target sequences was made possible by the linear nature of

RCA. Assuming a circular probe of 80 nucleotides and a replication rate of 1.4–

1.5 kb per minute it can be calculated that the circle amplification is as high as 1000fold per hour. Since the fluorescent decorator hybridizes with the RCA product, the

fluorescence intensity is directly proportional to the accumulation of amplification

product and can therefore be used to quantify the number of detected target sites.

The fluorescence signal was quantified for the droplet-encapsulated cells and compared to empty droplets. The calculated fluorescence intensity of LMP-1 and

EBNA-3, the single-copy genes, was about seven times lower than that of EBNA2IR, which has a known copy number of 7–13. Collectively, these results indicate

that this detection approach is capable of quantitative detection of the target sites.

The specificity of the method was again confirmed using the EBV-negative CCRFCEM cells, which did not produce a false positive signal (Fig. 10.3d).



5



Conclusions



The unique properties of PNA–DNA interaction open the door for a wide range of

opportunities in the field of DNA diagnostics. The sensitive and specific detection of

oncoviral DNA inserts demonstrates that PNA-assisted RCA is applicable to the convenient cells-in-flow format. The sequence specificity of PNA invasion and the linear

signal amplification of RCA make it possible to detect the smallest and most common

genetic variations, SNPs, and to quantify the number of target sites per cell using fluorescence intensity. This is a significant advantage over most cytogenetic techniques for

DNA detection, which are geared toward the detection of targets that are 1–2-kb long.



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Chapter 11



Sensor Systems with Magnetic

and Optomagnetic Readout of Rolling Circle

Amplification Products

Mikkel F. Hansen, Marco Donolato, Jeppe Fock, Mattias Strömberg,

Maria Strømme, and Peter Svedlindh



1  Introduction

This chapter describes the detection of products of a rolling circle amplification

(RCA)) process by use of magnetic nanoparticles (MNPs).

Most often, the RCA products are generated as the result of recognition of a

DNA or RNA target by a so-called padlock probe: first, hybridization of the ends of

a ≤90-nt-long linear oligonucleotide to a target nucleic acid sequence brings the

probe’s termini close to each other; next, the probe is circularized by an appropriate

ligase (thus forming a closed padlock), and finally the circular probe is employed as

a template for isothermal amplification via RCA (Nilsson et al. 1994). The target-­

unspecific “backbone” sequence of the padlock probe can include several

M.F. Hansen, Ph.D. (*) • J. Fock, Ph.D.

Department of Micro- and Nanotechnology, Technical University of Denmark,

DTU Nanotech, Building 345B, Kongens Lyngby 2800, Denmark

e-mail: Mikkel.hansen@nanotech.dtu.dk; jepf@nanotech.dtu.dk

M. Donolato, Ph.D.

Department of Micro- and Nanotechnology, Technical University of Denmark,

DTU Nanotech, Building 345 East, Kongens Lyngby 2800, Denmark

BluSense Diagnostics,

Symbion Bioscience Park, Fruebjergvej 3, Box 68, Copenhagen Ø 2100, Denmark

e-mail: marco@blusense-diagnostics.com

M. Strưmberg, Ph.D. • P. Svedlindh, Ph.D.

Division of Solid State Physics, Department of Engineering Sciences, Uppsala University,

The Ångström Laboratory, Box 534, Uppsala SE-751 21, Sweden

e-mail: Mattias.Stromberg@angstrom.uu.se; peter.svedlindh@angstrom.uu.se

M. Strømme, Ph.D.

Division of Nanotechnology and Functional Materials, Department of Engineering Sciences,

Uppsala University, The Ångstrưm Laboratory, Box 534, Uppsala SE-751 21, Sweden

e-mail: Maria.Stromme@Angstrom.uu.se

© Springer International Publishing Switzerland 2016

V.V. Demidov (ed.), Rolling Circle Amplification (RCA),

DOI 10.1007/978-3-319-42226-8_11



123



124



M.F. Hansen et al.



u­ ser-­chosen sequences as well as a restriction site for convenient detection of amplicons (Nilsson et al. 1994).

During RCA, the circularized padlock probes are copied to form a concatemer of

repeats of the sequence complementary to that of the padlock probe. Typically, the

RCA proceeds for 60 min, which results in about 1000 repeats of the monomer

sequence in each RCA amplicon (Banér et al. 1998). The sensitivity can be enhanced

by performing one or several cycles of a so-called circle-to-circle amplification

(C2CA), where the RCA product is restriction digested to form the template for a

new RCA (Dahl et al. 2004). The resulting single-stranded RCA products self-­

assemble into a compact DNA coil with a diameter around 1 μm (Banér et al. 1998).

The quantitation of RCA amplicons has commonly been based on visualization

of fluorescently labelled DNA coils in a confocal microscope or a laser scanner,

where such coils will appear as ~1-μm-diameter bright “blobs” (Jarvius et al. 2006;

Smolina et al. 2005). Although this approach is highly sensitive, as it is able to

detect sub-fM concentrations of RCA amplicons (Jarvius et al. 2006), it is only

applicable in a laboratory setting with expensive and specialized equipment.

Therefore, there is a need for alternative RCA-sensing principles that are more suitable for diagnostic applications.

Below, we present several different MNP-based approaches to RCA detection,

which were developed and tested by us towards this goal. Detection of RCA products using MNPs can be done either with untreated amplicons in the form of DNA

coils or with amplicons digested to individual repeating units. All employed readout

strategies are in a homogeneous (or volume-based assay) format, and they are based

on detection of changes in the rotational response of MNPs in suspension to an

applied oscillating magnetic field when detection probes attached to MNPs hybridize to RCA products.



2  Rotational Response of Magnetic Nanoparticles

The magnetic nanoparticles employed in our work are commercially available, and

they are typically so-called multi-core magnetic nanoparticles, where each particle

consists of several iron oxide nanocrystals encapsulated in an organic coating. The

particles have diameters in the range 50–250 nm, with a typical diameter being

100 nm. The particles have a remnant magnetic moment and will rotate to align this

moment along the direction of an applied magnetic field. The characteristic time

scale for the particles to reach equilibrium in an applied magnetic field depends on

their hydrodynamic volume, Vh, the viscosity of the liquid, η, and the thermal energy

kBT, where kB is Boltzmann’s constant and T is the absolute temperature. The process

is referred to as Brownian relaxation, and the response of an ensemble of particles

with identical sizes is characterized by the Brownian relaxation frequency

fB =





kBT

.

6phVh



(11.1)





11  Sensor Systems with Magnetic and Optomagnetic Readout of Rolling Circle…

Table 11.1 Brownian

relaxation frequencies fB

calculated for spherical

particles with hydrodynamic

diameters Dh in water

( h = 1mPas ) at room

temperature (T = 295  K)



Dh[nm]

50

100

250

1000



125

fB [Hz]

3300

413

26

0.4



Table 11.1 shows values of fB in water calculated for spherical particles with different hydrodynamic diameters Dh. A weak oscillating external magnetic field,

B ( t ) = B0 sin ( 2p ft ) , causes a dynamic magnetic response of an MNP ensemble.

This magnetic response may lag behind the applied magnetic field and may thus

show a negative phase shift, or equivalently, a positive phase lag ϕ with respect to

B(t). The time-dependent magnetic response can be written as

m ( t ) = mAC sin ( 2p ft - f ) = m¢ sin ( 2p ft ) - m² cos ( 2p ft ) with m¢ = mAC cos f and

m² = mAC sin f where both mAC and ϕ depend on f. Often, the response is more

conveniently described in the frequency domain in terms of the complex magnetic

susceptibility





c = c ¢ - ic ² = c ( cos f - i sin f )







(11.2)



where χ′ and χ″ are the in-phase and out-of-phase components of the susceptibility

and |χ| is the magnitude of the response to the applied magnetic field oscillation. The

complex susceptibility can conveniently be measured using lock-in detection techniques and the results can be presented either in terms of the magnitude and phase

or the in-phase and out-of-phase components of the signal. The two representations

are linked according to Eq. (11.2).

Figure  11.1a, b show curves of χ′, χ″ and ϕ calculated vs. frequency of the

applied magnetic field using the so-called Debye model (Debye 1929) for ensembles of particles with hydrodynamic diameters of 100 and 250 nm. The magnetic

response vs. frequency can be understood as follows:

On one hand, at frequencies much lower than fB, the MNP magnetic moments are

able to rotate to adjust their orientations while the field oscillates and thus they will

show a response that is in phase with the oscillating magnetic field ( f = 0° ). On the

other hand, at frequencies well above fB, the MNP magnetic moments are not able

to change orientation while the field changes and thus they show nearly no response.

At frequencies near fB, the MNPs are able to respond to the oscillating field, but their

response lags behind the oscillating magnetic field and a peak in the out-of-phase

magnetic response is observed at f = f B corresponding to a phase lag of f = 45° .

Thus, measurements of the complex magnetic response can be used to detect

changes in the distribution of hydrodynamic sizes of the particles. These changes

are most conveniently monitored in the out-of-phase component of the magnetic

response via changes of either the height or the position of the peak in the χ″ data.

Generally, the binding of small biological targets to MNPs is not easily detected

as the binding of these only results in a minor change of the hydrodynamic volume



126



M.F. Hansen et al.



Fig. 11.1  Signals vs. frequency f of the applied magnetic field calculated for particles with hydrodynamic diameters of 250 nm (dotted blue) and 100 nm (red) corresponding to Brownian relaxation frequencies fB1 and fB2, respectively. (a) Magnetic susceptibility with in-phase and out-of-phase

components χ′ and χ″, respectively. The χ″ signal peaks at f = f B . (b) Corresponding phase lag

ϕ of the magnetic response. (c) Optomagnetic signal with in-phase and out-of-phase components

V2′ and V2″. The V2″ signal crosses zero at f = f B and the V2′ signal shows a peak at a position

related to fB



of the MNPs. For example, the binding of targets resulting in a 10 nm increase of

the hydrodynamic diameter of 100 nm MNPs only yields a hydrodynamic volume

change of about 30 %. Due to the inevitable size distribution of particles and the

broad curve for a single MNP size in the χ″ data, it is not easy to resolve a small

change in MNP size with captured targets that would overlap with the curve from

free MNPs.

However, RCA products in the form of DNA coils have a size on the order of

1 μm. Thus, MNPs functionalized with capture oligonucleotides that bind to DNA

coils show an appreciable change in hydrodynamic size, which can easily be

detected as a turn-off (decrease) of the signal near fB for the free MNPs and/or as a

turn-on (increase) of a signal at low frequencies due to MNPs bound to DNA coils.

Several detection strategies for these effects are available that will be discussed

further in Sect. 4.

Recently, an optical approach for the readout of MNP dynamics has been

proposed and demonstrated (Donolato et al. 2015a). In this approach, the modulation of the intensity of transmitted laser light is measured in response to an

oscillating magnetic field applied either along the light path or perpendicular to

it. The method is based on the fact that the MNPs are not spherical and have

established a link between their optical and magnetic anisotropies, for example,

when the particles are elongated and have a magnetic moment along their long

axis. Thus, the intensity of the transmitted light depends on whether the particles are randomly oriented or aligned along the applied field. We have shown

that the complex second harmonic of the transmitted light signal with respect to

the magnetic field excitation can be written in terms of the phase lag ϕ of the

magnetic response as





V2 = V2¢ + iV2² = V2 ( sin ( 2f ) + i cos ( 2f ) )







(11.3)



11  Sensor Systems with Magnetic and Optomagnetic Readout of Rolling Circle…



127



and for low magnetic fields we have argued that V2 is linked to the magnetic susceptibility as



( )( c )

é( c ) - ( c ) ù

ëê

ûú



V2¢ = VAC c ¢





V2² = VAC



¢



2



²



²



(11.4)



2







where VAC is a constant that depends on the measurement geometry, the MNP properties, and the magnetic field amplitude (Donolato et al. 2015a). Figure 11.1c shows

the in-phase and out-of-phase second harmonic optomagnetic response corresponding to the magnetic susceptibility curves in Fig. 11.1a calculated from Eq. (11.4)

with VAC = 1 . The values of the Brownian relaxation frequencies are observed to

correspond to the points where the V2″ signal crosses zero, but it is also observed that

the in-phase component of the second harmonic response, V2′, shows a peak-­like

feature, which is related to the Brownian relaxation frequency (but not equal to it).

The change of hydrodynamic size of the MNPs due to binding to DNA coils can

conveniently be detected as a turn-off of the peak in the V2′ data or via measurements

of the change in the phase lag ϕ of the magnetic signal. Both types of signals can be

obtained via phase-sensitive (or lock-in) detection, which measures both the magnitude and the phase of the signal with respect to the magnetic field excitation.



3  Instrumentation

In this section, we will give an overview of the electronic and optoelectronic sensing

devices conveniently used by us to measure the RCA-product-induced dynamic

magnetic response of MNPs in suspension. Each instrument (see Fig. 11.2) will be

described with a focus on operation, practicality, and portability.



3.1  Tabletop AC Susceptometer

After proof-of-principle studies performed with a sophisticated, bulky, and costly

superconducting quantum interference device (SQUID, Fig. 11.2a; Strömberg et al.

2008a, b), the AC susceptometer DynoMag (Acreo Swedish ICT) has been used

(Fig. 11.2b). This portable, easy-to-use instrument costs significantly less than the

SQUID magnetometer, it has the size of a personal computer, and it operates at

room temperature. The DynoMag susceptometer measures inductively the complex

magnetic response of a sample to a small AC magnetic field excitation with a fixed

amplitude of 0.5 mT generated inside an excitation coil in the device.

Also important is that the DynoMag susceptometer requires a sample volume of

a few hundred microliters. It typically operates at frequencies in the range between



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