DNAzyme conjugated nanomaterials for biosensing applications

DNAzyme conjugated gold nanoparticles in biosensing

There are some unique properties for gold nanoparticles (AuNPs), such as optical properties, distance-dependent surface plasmon properties, and AuNPs are essentially nontoxic, biocompatible, easily functionalized with a variety of ligands, and relatively stable in solution. Among these, distance-dependent surface plasmon was the most used property when constructing AuNPs-based biosensors. The color of AuNPs solution turns from red to purple with the aggregation of the dispersed AuNPs. According to this principle, a series of colorimetric sensors based on DNAzyme assembly-induced gold nanoparticles aggregation were designed (Liu and Lu 2003, 2004a,b, 2005, Lee et al. 2008, Wang et al. 2010, Luo et al. 2012b, Zagorovsky and Chan 2013). Some of these colorimetric sensors are used for sensitive and selective detection and quantification of Pb (II). For example, employing Pb (II)-dependent 8–17 DNAzyme, Lu et al. developed several colorimetric AuNPs sensors (Liu and Lu 2003, 2004a,b, 2005). As shown in Figure 2A, the substrate (17 DS) extends 12 bases at double ends of the strand, and assemble, on the surface of gold nanoparticles. The nanoparticles are aligned in a tail-to-tail manner (Figure 2B). In addition, an invasive DNA was introduced to improve the reaction rate. Without Pb²⁺, the substrate cannot be incised by the enzyme and the solution stays blue. Once the Pb²⁺ was added, the enzyme was catalyzed to incise the substrate, which induces the aggregation of gold nanoparticles and subsequently the color change of the solution from blue to red. By taking advantages of this colorimetric sensor, we can clearly determine whether Pb²⁺ exists or not by the naked eye the detection limit of 1 μm. Furthermore, considering that unfolded single-stranded DNA would be adsorbed by the citrate protected AuNPs while double-stranded DNA would not, Wang and co-workers (Wei et al. 2008) designed another kind of DNAzyme-based colorimetric sensor and realized the simple, rapid, cost-effective, and sensitive detection of Pb²⁺. As we know, with the exception of, the spread of infections diseases, heavy metals have caused significant loss to the economies and human health, and to detect the pathogens early and determine appropriate is an effective method of control. Quantitative PCR (qPCR) is one of the widely used methods to detect pathogen DNAs, and these DNAs act as important biomarkers of pathogen. However, qPCR is limited for use in medical laboratories because it requires expensive equipment and highly trained personnel. To develop cheaper and simpler DNA detection methods, Chan and co-workers (Zagorovsky and Chan 2013) combined DNAzyme with AuNPs and thus designed a simple point-of-care diagnostic technique for the detection of infectious pathogens. They took the advantage of a multicomponent nucleic acid enzyme (MNAzyme), and successfully provided a sensitivity strategy to detect genetic targets from bacteria under a target concentration of 50 pm without purification and separation steps. Also, other colorimetric agents are available to be introduced into metal nanoparticle-based colorimetric reporters. Wang and co-workers (Zhu et al. 2013) reported a visible multi-digit DNA keypad lock based on silver microspheres and split G-quadruplex DNAzyme, with several advantages such as being easily recognized by the naked eye, low-cost and environment friendly, scalability and flexibility.

Figure 2

Pb²⁺-directed assembly of gold nanoparticles by the DNAzyme when nanoparticles are aligned in a head-to-tail (A) or a tail-to-tail manner (B). Reprinted with permission from: Liu and Lu 2005. Copyright 2005, American Chemical Society. (C) Amplified chemiluminescence detection of DNA using DNAzyme-functionalized AuNPs. Reprinted with permission from: Willner et al. (2004). Copyright 2004, American Chemical Society.

Recently, researchers studied the strong signal enhancement effect of AuNPs in different biosensing systems to substantially improve the performance and sensitivity. Ye and co-workers (Yin et al. 2010) used a “nanoparticle enhancement” approach to realize accurate and rapid detection of Cu²⁺ and Pb²⁺ ions based on fluorescence anisotropy assay in aqueous medium at room temperature. The substrate strand, which was labeled with 6-fluorescein-CE phosphoramidite (FAM) at the 3′end and thiol group at the 5′end, could be immobilized onto the surface of AuNP via an S-Au bond. After the hybridization of DNAzyme with substrate, the FAM fluorophore on the “big” AuNP-mediated substrate and enzyme complexes showed a stable anisotropy signal. In the presence of metal ions, the released fluorophore with cleavaged substrate displayed rapid rotation, which depolarized the light and showed a low anisotropy signal. Based on these findings and results, a new method for rapid and easy detection of toxic metal ions in environmental samples has been put forward. Willner’s group (Niazov et al. 2004) used AuNPs as carriers for the amplified detection of DNA or telomerase activity. In their design, luminol/G-quadruplexes were used to produce biochemiluminescence as a readout signal. This method enabled the detection of telomerase activity originating from 1000 HeLa cells.

Another unique optical property of AuNPs is their fluorescence quenching ability. By using AuNPs as quenchers, Liu and co-workers (Wang et al. 2013) developed a novel turn-on fluorescence biosenser based on GR-5 DNAzyme for rapid, simple, sensitive and selective Pb²⁺ detection. When the DNAzyme was modified on the surface of AuNP, the FAM labeled GR-5 DNAzyme was close enough to the AuNP for the Au the fluorescence signal to be quenched. In the presence of Pb²⁺ ions, the substrate strand was cleaved by the DNAzyme, which led to short FAM-linked oligonucleotide release from the AuNP and the fluorescence intensity was greatly recovered. This method exhibited a high sensitivity toward Pb²⁺ with a detection limit of 250 pM. In addition, the sensing strategy used to determinate Pb²⁺ ions in lake water samples obtained a satisfying outcome. Although many DNAzyme-based fluorescent Pb²⁺ sensors have been reported, most of them suffered from incomplete fluorescence quenching. Thus annealing DNAzymes and substrates as well as removing the uncoupled substrates was required. To overcome this problem, Chung and co-workers (Joong Hyun Kim et al. 2011) immobilized the substrates on AuNPs through thiol Linkages and acquired almost complete fluorescence quenching. Although DNAzymes have shown great promise as a general platform for detecting metal ions in the extracellular environment, however, intracellular application of DNAzyme-based sensors have not been reported due to the difficulty of protecting DNAzyme from enzymatic degradation and delivering DNAzyme into cells. To make a breakthrough, Lu and co-workers (Wu et al. 2013) chose AuNPs as the carriers to realize intracellular delivery of DNAzymes, using modification with phosphorothioate linkage increase their stability. Because the AuNP-DNA conjugate has many desirable properties including stability in serum, the ability to enter cells without use of transfection agents, a much larger DNA loading efficiency, and increased resistance to enzymatic degradation. This method provides a simple and general platform to convert DNAzymes into intracellular probes for metal ions detection.

Electro-chemical methods have also been employed to design DNAzyme-based biosensors for the detection of metal ions due to their remarkable characteristics, such as simple instrumentation, high sensitivity, promising response speed, and low production cost. Shao and co-workers (Shen et al. 2008) developed an electrochemical DNAzyme sensor for sensitive and selective detection of Pb²⁺. Amplified by DNA-Au bio-bar codes, which have larger surface areas to bind a larger number of short DNA sequences, the electrochemical DNAzyme biosensor exhibits ultrasensitive detection ability for Pb²⁺. This kind of electrochemical DNAzyme biosensor provides a platform to analyze a series of small molecules and metal ions. Guo and coworkers (Li et al. 2011) developed a DNAzyme-based electrochemical method for real-time detection of L-histidine. Functionalized with a redox probe (ferrocene) at the 5′end, the DNAzyme strand was assembled on the surface of an AuNP (15 nm), which was immobilized on the gold electrode surface to improve sensitivity. Via a thiol-gold interaction, the substrate strand was hybridized to the DNAzyme strand, which prohibited any contact between ferrocene and the electrode. In the presence of L-histidine, the substrate was cleaved and released, which made the enzyme strand more flexible and facilitated the electrochemical communication between the electrode and the redox probe. This electrochemical sensor produced an electrochemical signal proportional to the concentration of L-histidine present with a detection limit of 0.1 pm. Tang and co-workers (Xu et al. 2013) integrated difunctional DNA-nanogold (AuNP) dendrimer with a hemin/G-quadruplex as the nanotag to detect nucleic acid and achieved an amplified electronic signal in situ by coupling high-efficiency DNAzyme with the intercalated methylene blue (MB).

Surface-enhanced Raman scattering (SERS) has been widely used for biological sensing. It was also reported that AuNPs with size >20 nm could produce an electromagnetic field around the gold surface and provide strong Raman enhancement. Zhang and co-workers (Sun et al. 2011) utilized a nanoscale DNAzyme-Au dendrimer as a signal amplifier and designed an ultra-sensitive SERS biosensor (Figure 3). The novel and universal DNA-Au dendrimer-based sensing system provides a new platform for the amplified detection of various targets such as small molecules and metal ions. Miao et al. (2011) combined AuNPs with DNAzyme and designed a dynamic light scattering sensor for Pb²⁺ detection with a detection limit of 35 pM. Later, Zhang and co-workers (Ye et al. 2013) demonstrated a novel surface-enhanced Raman scattering (SERS) method to detect L-histidine based on DNAzyme. This sensing system afforded high sensitivity for L-histidine with a detection limit of 0.56 nm through a signal-amplifier strategy of a target recycling cascade.

Figure 3

(A) Schematic of the amplified sensing strategy of the DNA-Au dendrimer-based SERS catalytic beacon for Pb²⁺. Rox stands for X-rhodamine, and MCH denotes 6-mercaptohexanol. (B) Sensitivity of the DNA-Au dendrimer-based SERS DNAzyme biosensor for Pb²⁺ detection: (a) SERS spectra of the DNAzyme-based sensing interface with varying concentrations of Pb²⁺; (b) Calibration curve of the SERS DNAzyme Pb²⁺ sensor. Reprinted with with permission from: Sun et al. 2011. Copyright 2011, Royal Society of Chemistry.

DNAzyme conjugated carbon nanomaterials in biosensing

As one of the most plentiful elements in nature, carbon forms numerous crystalline structures including fullerenes, graphene, carbon nanotubes, graphene nanoribbons, and diamond clusters, etc. (Terrones 2010). Carbon nanomaterials have some excellent properties like good biocompatibility, nontoxicity and stability, and, different kinds of carbon nanomaterials have special features individually. As a result, carbon nanomaterials were widely applied in chemistry and biology. In this review, we mainly discuss the biosensing applications of DNAzyme-conjugated graphene oxide and carbon nanotubes (CNTs).

DNAzyme conjugated graphene oxide for biosensing applications

Graphene (individual sp²-hybridized carbon sheets), is a novel one-atom-thick, two-dimensional graphitic carbon system with excellent features, such as extraordinary electronic, optical and thermal properties (Geim and Novoselov 2007, Li et al. 2008). And, graphene is also a good energy acceptor in energy transfer. However, graphene has poor water solubility which is difficult to modify, which has limited its application. In recent decades, due to its unique characteristics including good water dispersibility, facile surface modification and high mechanical strength, graphene oxide (GO) has attracted increasing interest in biological applications, such as DNA analysis (Lu et al. 2010), enzyme activity analysis (Jang et al. 2010), protein assays (Chang et al. 2010) and drug delivery (Liu et al. 2008). For the most part, graphene oxide was predicted through theoretical calculations to be a super quencher with long range nanoscale energy transfer properties. Zhang and co-workers (Zhao et al. 2011) designed a GO-DNAzyme based biosensor for amplified fluorescence detection of Pb²⁺ (Figure 4). In their design, GO acted as a superquencher, while the FAM-labeled DNAzyme-substrate complex acted as both signal reporter and target receptor. DNAzyme could be absorbed on GO through electrostatic interaction and the π–π stacking between the rings of the nucleotide bases of the ssDNA and GO. Due to the super fluorescence quenching efficiency of GO, the “turn-on” biosensor exhibits a high sensitivity toward Pb²⁺ with a detection limit of 300 pM. Wen et al. (2011) utilized the interaction difference between GO with single stranded (ss-) and double stranded (ds-) DNA leading to different quenching of fluorescence and developed a similar method to detect Pb²⁺. A similar chemiluminescence biosensor for DNA detection was reported by He and co-workers (Luo et al. 2012a), which demonstrated that graphene oxide would greatly inhibit the horseradish peroxidase-mimicking DNAzyme.

Figure 4

(A): (a) Secondary structure of the GR-5 DNAzyme hybridized with the FAM-labeled substrate strand; (b) secondary structure of the 8–17 DNAzyme hybridized with the FAM-labeled substrate strand; (c) schematic illustration of the DNAzyme_GO based fluorescence sensor for Pb²⁺. (B): (a) Fluorescence emission spectra of the sensing system on exposure to Pb²⁺ solutions of different concentrations and then mixed with GO; (b) calibration curve of the sensing system for Pb²⁺. The curve was plotted with thefluorescence enhancement vs Pb²⁺ concentration. Inset shows the linear responses at low Pb²⁺ concentrations. Reprinted with permission from: Zhao et al. 2011, copyright 2011, American Chemical Society.

Huang and co-workers (Yu et al. 2013) demonstrated that introducing graphene oxide (GO) could effectively enhance the fluorescence anisotropy (FA) of fluorophore. Thus, they developed a highly sensitive and selective method for the detection of metal ions through an anisotropy DNAzyme-based strategy. Two DNA strands, including DNAzyme (Cu-Enz) and substrate DNA labeled with TAMRA in 3′end, were designed to assemble with GO through electrostatic interaction and the π–π stacking between the bases of unpaired sequence and GO. According to the Perrin equation, the rotation of TAMRA obtained high anisotropy. In the presence of Cu²⁺, the TAMRA-labeled DNA substrate strand was cleaved by DNAzyme and released from the GO surface because of the much weaker π–π stacking interaction between GO and short ssDNA. As a result, TAMRA rotated at a rate commensurate with its small size and provided the most dramatic decreases in FA.

DNAzyme conjugated carbon nanotubes for biosensing applications

Carbon nanotubes (CNTs), also named rolled graphene sheets, can be classified into single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) (Ajayan 1999). CNTs have unique properties with their hollow monolithic structure with distinct inner and outer surfaces: the outer surface can be modified with different molecules for functionalization and the inner voids can be filled with species ranging from large proteins to small molecules (Mitchell et al. 2002). CNTs have the same properties as graphene with different adsorption abilities between ss- and dsDNA and super fluorescence quenching efficiency. It has been proved that the water-solubility of CNTs can be improved by conjugation with DNA, so DNA, especially DNAzyme-conjugated CNTs, have been used widely in constructing nanodevices, for example, biosensors.

Jonathan S. Dordick et al. (Yim et al. 2005) developed an effective strategy for highly active and stable DNAzyme-carbon nanotube hybrids. As shown in Figure 5, streptavidin (STV) was first attached covalently onto MWNTs through stable amide linkages with the help of two crosslinking agents EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide). Then biotinylated DNAzyme was allowed to bind to the STV-MWNT to yield the water soluble DNAzyme-MWNT conjugates. Finally, FAM-labeled substrate DNA was hybridized with DNAzyme and the DNAzyme-MWNTs conjugates were demonstrated to be highly active. Thus, the catalytic activity and selectivity of these conjugates might enable the directed assembly of nanomaterials into functional 3D architectures and the delivery capability of nanotubes (Figure 5). Zhong and co-workers (Yao et al. 2011) reported a sensitive and simple assay for combining DNAzyme with single-walled carbon nanotubes for the detection of Pb²⁺ in aqueous solutions. The substrate strand was labeled with Cy3. In the presence of Pb²⁺, the DNAzyme cleaved the fluorophore-labeled DNA substrate. The single-stranded product was released and adsorbed onto SWCNT, leading to Cy3 quenching. Utilizing the noncovalent assembly of DNA and single-walled carbon nanotubes (SWNTs), Wang and co-workers (Ding et al. 2011) developed a novel homogenous method for the detection of genomic DNA and Staphylococcus aureus based on the self-assembled DNAzyme probes and SWNT. When the target DNA and hemin existed in the solution, the detection probe hybridized with target DNA, kept away from SWNTs and formed a self-assemble complex. Then the DNAzyme catalyzed 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^2–) and produced a colored product that could be recognized by the naked eye.

Figure 5

(A): (a) Sequence of the 17E DNAzyme and schematic showing the procedure for immobilizing it onto MWNTs; (b) sequence of 6-carboxyfluorescein (FAM)-bound substrate DNA, which contains a single embedded ribonucleotide (cleavage site), along with a cartoon depicting its hybridization with DNAzyme. (B) Catalytic activity of DNAzyme-MWNT conjugates: (a) initial rate of cleavage of substrate DNA by DNAzyme-MWNT conjugates in the presence of 0.15 mm Pb²⁺; (b) confirmation of multiple turnovers by DNAzyme catalysis initiated with 0.15 mm Pb²⁺ at room temperature. Reprinted with permission from: Yim et al. 2005. Copyright 2005, American Chemical Society.

DNAzyme conjugated magnetic nanomaterials in biosensing

Due to their unique advantages, nanoscale magnetic materials provides many exciting opportunities in biology and biomedical applications, such as sensing, imaging and remote heating (Kalambur et al. 2005). High throughput separation capability is one of the most outstanding features of magnetic nanoparticles, which is usually used for sensitive detection of targets of interest. Lu and co-workers (Xiang and Lu 2013) reported a general methodology for metal ion detection using a personal glucometer with an invasive DNA approach through streptavidin-coated magnetic nanobeads (Figure 6). Part of the substrate strand of the DNAzyme acted as invasive DNA for DNA-invertase conjugates, while invertase could hydrolyze sucrose into glucose. In the presence of catalytic factors (also targets), a substrate strand was cleaved, and the cleaved DNA competed with DNA-invertase conjugates to hybridize with the biotinylated DNA. This bounded on streptavidin-coated MBs, and released the DNA-invertase conjugates into the solution. After magnetic separation, DNA-invertase conjugates transformed sucrose into glucose, which was measured by a PGM to determine the cofactors concentration in the samples. Nie et al. (2012) developed a sensitive and selective DNAzyme-based flow cytometric method to detect Pb²⁺. This is the first report about a flow cytometric-based DNAzyme method for metal ion detection with a detection limit of 0.6 nm. In order to get a simple sensing method for detection of metal ions with lower detection limit, Zeng and co-workers (Ge et al. 2013) developed an enzyme-free and label-free assay for copper (II) ion detection based on self-assembled DNA concatamers and enabled a quantitative detection limit of Cu²⁺ as low as 12.8 pM. Magnetic nanoparticles can also work as amplifiers. Using magnetic nanoparticles for target separation and signal amplification, Wang and co-workers (Du et al. 2011) developed a G-quadruplex-based DNAzyme sensor for colorimetric detection of cocaine.

Figure 6

(A) Pb²⁺-induced and UO₂²⁺ -induced cleavage of the DNA substrates by the corresponding DNAzyme. Both reactions yield the cleaved ssDNA product (red) as invasive DNA and release of DNA-invertase conjugates from MBs by the invasive DNA. (B) The invasion and release steps, followed by the conversion of sucrose into glucose by the released DNA-invertase conjugates for PGM measurement. Reprinted with permission from: Xiang and Lu 2013. Copyright 2013, Royal Society of Chemistry.

Rolling circle amplification (RCA) has been proved to be a promising biological signal amplification strategy. Li and co-workers (Tang et al. 2012) developed a novel aptamer-functionalized microbead-based protein detection assay that integrates RCA with a DNAzyme-catalyzed colorimetric reaction. With the dual-amplification strategy, this design could achieve good detection performance when used in protein marker detection in a serum-containing medium, which when compared with traditional assays such as ELISA, means a detection limit of 0.2 pg/ml. A similar design was also reported by Zhang and co-workers (Bi et al. 2010) for thrombin detection.

DNAzyme conjugated quantum dots in biosensing

Due to their unique optical properties, quantum dots (QDs) have been researched widely as signal labels or electrochemical tracers for sensor construction. Generally, QDs were modified with DNA as a quencher for the fluorescence resonance energy transfer (FRET), electron transfer (ET) or chemiluminescence resonance energy transfer (CRET) to analyze DNA or antigen-antibody complexes, such as intercalatores or AuNPs (Freeman et al. 2013) (Figure 7). The emission spectral range of QDs is in the near-IR, which is advantageous for cell imaging and biosensing. In addition, QDs have many remarkable properties, such as high photoluminescence intensity, high quantum yields, broad ultraviolet (UV) excitation and resistance to photo- and chemical-degradation. To enhance their quantum yield, these QD cores are often overcoated with a layer of inorganic material (e.g., ZnS) (Hu et al. 2011). These excellent properties make them the ideal platform for biosensing and hence the combination of DNAzyme and QDs has attracted a lot of interest.

Figure 7

Schematic representation of different QD-based sensing configurations using (A) luminescence, (B) electron transfer, ET, (C) FRET, or (D) photocurrent generation as readout signals. Reprinted with permission from: Freeman et al. 2013. Copyright 2013, American Chemical Society.

Sharon et al. (2010) proposed a design in which the horseradish peroxidase (HRP)-mimicking hemin/G-quadruplex DNAzyme as a catalytic label was conjugated with CdSe/ZnS QDs to develop a DNA sensor for the detection of DNA or aptamer-substrate complexes. The luminescence of CdSe/ZnS QDs is quenched via ET by hemin/G-quadruplex connected with the nanoparticles, which made it possible to develop different optical sensing platforms. Later, they reported a sensor for the detection of aptamer substrate complexes, metal ions (Hg²⁺) (Freeman et al. 2011), and DNA via CRET between self-assembled hemin/G-quadruplexes nanostructures and CdSe/ZnS QDs. There are several advantages for the confinement of the CRET reaction to the hemin/G-quadruplex DNAzyme-QDs conjugates, such as: 1) due to diffusional hemin, chemiluminescent background signals were eliminated; 2) through different sized QDs and variable compositions, multiplex analyses using a common internal light source became possible. At the same time, they reported another CRET aptamer sensor by using catalytic hemin/G-quadruplexes and CdSe/ZnS semiconductor QDs, with detection limits of 200 pm for thrombin, and 10 mm for ATP (Liu et al. 2011). Fan and co-workers (Wu et al. 2010) developed nanosensors for highly sensitive multiplexed heavy metal detection using QD-labeled DNAzymes via FRET. In their design, quencher-labeled DNAzymes were conjugated to the surface of carboxyl-silanized QDs and the fluorescence of the QDs was therefore efficiently quenched due to the close proximity. In the presence of metal ions, the fluorescence of QDs recovered due to the cleavage of DNAzymes. By combining the highly stable silanized QDs with high ion-specific DNAzymes, the QD-DNAzyme sensors realized the possibility of ultrasensitive, selective and multiplexed detection of heavy metal ions in liquid.