Blu-ray based optomagnetic aptasensor for detection of small molecules

Highlights

• A small molecule (ATP) is quantified using an aptamer-based optomagnetic biosensor.

• ATP inhibits hybridization and clustering of aptamer and DNA magnetic nanoparticles.

• Optomagnetic readout determines clustering state from size-selective measurements.

• Readout uses low-cost components (Blu-ray pick-up unit, mirror, electromagnets).

Abstract

This paper describes an aptamer-based optomagnetic biosensor for detection of a small molecule based on target binding-induced inhibition of magnetic nanoparticle (MNP) clustering. For the detection of a target small molecule, two mutually exclusive binding reactions (aptamer-target binding and aptamer-DNA linker hybridization) are designed. An aptamer specific to the target and a DNA linker complementary to a part of the aptamer sequence are immobilized onto separate MNPs. Hybridization of the DNA linker and the aptamer induces formation of MNP clusters. The target-to-aptamer binding on MNPs prior to the addition of linker-functionalized MNPs significantly hinders the hybridization reaction, thus reducing the degree of MNP clustering. The clustering state, which is thus related to the target concentration, is then quantitatively determined by an optomagnetic readout technique that provides the hydrodynamic size distribution of MNPs and their clusters. A commercial Blu-ray optical pickup unit is used for optical signal acquisition, which enables the establishment of a low-cost and miniaturized biosensing platform. Experimental results show that the degree of MNP clustering correlates well with the concentration of a target small molecule, adenosine triphosphate (ATP) in this work, in the range between 10 µM and 10 mM. This successful proof-of-concept indicates that our optomagnetic aptasensor can be further developed as a low-cost biosensing platform for detection of small molecule biomarkers in an out-of-lab setting.

Introduction

Magnetic nanoparticles (MNPs) have been widely used in biosensing for detection of various biomolecules such as nucleic acids (Go¨ransson et al., 2010, Josephson et al., 2001, Liong et al., 2013, Zhang et al., 2013b), proteins (Aurich et al., 2006, Liang et al., 2011, Ranzoni et al., 2012, Zhang et al., 2013a), enzymes (Bamrungsap et al., 2011), cells (Herr et al., 2006) and small molecules (Sun et al., 2006, Wu et al., 2011, Yigit et al., 2007, Zhang et al., 2009). Due to the low magnetic susceptibility of biological compounds, the manipulation and detection of MNPs in biological media are not susceptible to interference from magnetic background, and this allows for the efficient and sensitive detection of biomolecules without the need for sample purification steps.

MNP-based detection of biomolecules has been applied to both surface-based (Bruls et al., 2009, Mani et al., 2009, Nash et al., 2012, Tekin et al., 2013, Wang et al., 2010, Wang and Gan, 2009) and volume-based assays (Aurich et al., 2006, Bamrungsap et al., 2011, Chun et al., 2011, Go¨ransson et al., 2010, Josephson et al., 2001, Liang et al., 2011, Liong et al., 2013, Ranzoni et al., 2012, Sun et al., 2006, Zhang et al., 2013a). The latter typically employs sandwich binding-induced clustering where a molecule of interest is bound to multiple receptors tethered onto MNP surfaces, resulting in the agglutination of MNPs. Accordingly, the addition of a target triggers clustering of MNPs, leading to changes in the size and the number of agglomerated particles with the analyte concentration, which is referred to as a clustering assays (Aurich et al., 2006, Baudry et al., 2006, Castañeda et al., 2007, Go¨ransson et al., 2010, Josephson et al., 2001, Koh et al., 2009, Liang et al., 2011, Ling et al., 2010, Perez et al., 2002, Ranzoni et al., 2012, Zhang et al., 2013a). Such a volume-based MNP clustering assay is particularly appealing since it allows simple mix-and-read type measurements and reduced reaction time by use of external magnetic fields (Baudry et al., 2006).

Despite these advantages, the extensive use of MNP clustering assays has been impeded by the following limitations:

First, the typical clustering assay is applicable only to a target molecule with multiple binding sites, but not feasible for small molecules due to their limited number of binding sites. Therefore, target binding-induced cluster disassembly strategies have been pursued using a competitive binding assay format (Sun et al., 2006) or structure-switching signaling aptamers (Wu et al., 2011, Yigit et al., 2007, Zhang et al., 2009). However, in the competition-based approach, the formation of target-functionalized MNPs via a covalent bond is not always viable because of lack of functional groups within such small molecules, and the binding affinities of aptamers in the structure-switching approach are frequently reduced by competition, blocking (Nutiu and Li, 2003) and allosteric inhibition (Ricci et al., 2012) effects, thus lowering the detection sensitivity.

Second, current technologies to detect MNP clusters typically measure properties such as transverse relaxation time (Bamrungsap et al., 2011, Eberbeck et al., 2009, Kaittanis et al., 2011, Liang et al., 2011, Ling et al., 2010, Min et al., 2012, Perez et al., 2002), total sample magnetization (Michael et al., 2012), or light scattering (Chun et al., 2011, Michael et al., 2012). However, since these methodologies do not resolve signals with respect to the distribution of particle sizes, quantitative measurements of clusters in a mixture are readily interfered by background signals from a large amount of single MNPs or clusters of different sizes, thus lowering the accuracy and sensitivity.

Third, existing readout techniques require bulky and sophisticated electronic equipment such as nuclear magnetic resonance (NMR) spectroscopy, magnetometers equipped with accurate temperature control, or dynamic light scattering equipment, which in turn limit their applicability in an out-of-lab setting.

In this study, we develop an aptamer-based MNP clustering biosensor for detection of small molecules via particle size-selective measurements on a compact, simple, and inexpensive optomagnetic readout system. A conventional inhibition assay configuration is employed (Crowther, 2000), where two types of MNPs are prepared, one tagged with an aptamer (apt-MNPs), a single stranded oligonucleotide receptor (Ellington and Szostak, 1990), and the other with a sequence partially complementary to the aptamer (linker-MNPs), called a DNA linker. When a sample containing a target small molecule is incubated with apt-MNPs prior to the addition of linker-MNPs, the aptamer is folded into a three-dimensional structure stabilized by the target, referred to as adaptive binding (Da Costa et al., 2013, Gilbert et al., 2009, Hermann and Patel, 2000). From a thermodynamic viewpoint, such a stabilization implies that in the presence of its cognate target, the overall free energy of the hybridization reaction between an aptamer and its complementary DNA sequence becomes less negative (Da Costa et al., 2013). Thus, the small target molecule behaves like an inhibitor, and the resulting affinity constant K_eq′ of the aptamer for its complementary strand (DNA linker) is reduced and can be expressed as K_eq′=K_eq/(1+[T]×K_a), where [T] is the target concentration and K_a is the affinity constant for the aptamer-target binding. Consequently, since MNP clusters are formed by the aptamer-linker hybridization reaction, the degree of MNP clustering decreases with increasing target concentration. This target binding-induced particle clustering inhibition approach facilitates the detection of diverse small molecules by designing the linker strands to hybridize with known binding sequences of aptamers.

To determine the degree of MNP clustering, i.e., the cluster sizes and respective amounts, we implemented an optomagnetic readout system that collects transmitted light, which is modulated by rotating MNPs under the actuation of an external magnetic field (Donolato et al., 2015a). The optical signal is found to be closely related to the rotational dynamics of MNPs, which is linked to their hydrodynamic sizes. As a result, this optomagnetic measurement enables us to effectively detect optical signals with respect to cluster sizes, allowing an accurate and quantitative determination of the clustering state.

Furthermore, to simplify and miniaturize the readout platform, we utilize a Blu-ray optical pickup unit (OPU) that incorporates a laser diode and a photo detector costing less than 10$ (Donolato et al., 2015b). By integrating such a compact and reliable optical element with a cuvette for simple sample loading (Fig. S1), we establish a robust and miniaturized optomagnetic readout system.

In this proof-of-concept study, adenosine triphosphate (ATP) is chosen as a target small molecule. ATP is an intracellular energy transporter that regulates cell metabolism and signaling pathways. ATP is also used as an important indicator of cell viability (Leist et al., 1997) and its intracellular concentration ranges from 0.1 to 3 mM (Traut, 1994). Thus, it is important to detect ATP in this range for clinical and biomedical research. Combining the aptamer-based inhibitive MNP clustering strategy with size-selective measurements on a low-cost and miniaturized platform, we demonstrate in this proof-of-concept study that our optomagnetic aptasensor can achieve the detection of ATP in the concentration range from 10 µM to 10 mM.