Introduction

Molecular beacons (MBs), first proposed by Tyagi and Kramer [1] in 1996, are hairpin-structured probes (Fig. 1) which have been widely used in many areas, such as the detection of PCR products, mutational analysis, clinical diagnosis, genotyping, and allele discrimination [2–8]. The hairpin structures of MBs resemble the hairpin secondary structures commonly found in RNA, which lead to the unique properties of the probe. Briefly, a MB is an oligonucleotide that contains a photoluminescent species (PLS) and a quencher at different ends of the strand.Footnote 1 The MB has two distinctive parts: the loop portion, which is the probe part and is designed complementary to a desired target nucleic acid sequence, and the stem, which is formed of two self-complementary regions composed of five to six nucleotides at opposite ends of the strand. In the absence of the target, the complementary parts of the stem hybridize together, prompting the formation of a hairpin structure (loop–stem), bringing the PLS and the quencher into close proximity. Owing to the close proximity enforced structurally by the stem, the quencher deactivates the PLS excited state generally by energy transfer or collisional quenching, resulting in a strong quenching of the photoluminescence. However, after hybridization with the target, the MB changes its conformation, forcing the PLS and quencher far apart and resulting in restoration of the MB photoluminescence. This selective conformational change permits the observation of photoluminescence from the PLS in principle, only after the probe has selectively hybridized to the target [9, 10]. The first reported MB consisted of a five-base-pair stem and an 18-base loop section with 5-(2’-aminoethyl)aminonaphthalene-1-sulfonic acid as the PLS at the 5’ end and 4-(4’-dimethylaminophenylazo)benzoic acid (DABCYL) at the 3’ end acting as the quencher [1].

Fig. 1
figure 1

The principle of operation of a molecular beacon (MB). When the MB assumes a hairpin structure, the photoluminescence of the photoluminescent species (PLS) is quenched by the close proximity of the quencher (Q). Hybridization with the DNA target complementary to the loop sequence produces a conformational change that separate the PLS and the quencher, allowing photoluminescence

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MB probes have high specificity toward single-base mismatch; however, their sensitivity (defined as the ratio of the photoluminescence in the open versus the closed form; signal-to-background ratio) is limited in many cases owing to incomplete quenching in the closed form. Making better MBs involves improving the quenching efficiency, which is related to developing new PLS or quenchers, as well as innovative PLS and PLS–quencher systems [11].

A recent advancement in MB technology involves the use of hybrid materials for DNA and RNA detection [12]. Hybrid materials bring several advantages in improving the sensitivity of MBs, and many different probes have been developed [13–15]. The good recognition properties of MBs, combined with the unique optical properties of inorganic materials, make these composite materials promising for use in the field of bioanalysis [16]. Heavy-metal complexes, nanocrystals, pyrene compounds, and other materials with excellent photophysical properties have been applied as PLS of MBs. Nanoparticles, nanowires, graphene, metal films, and many other materials have also been introduced to quench photoluminescence. Combined with a variety of detection approaches, MBs have become a powerful tool for real-time detection of single-stranded DNA and messenger RNA (mRNA) in vitro and in vivo. Optimized MBs have subnanomolar detection limits, and a high level of simultaneous detection has been obtained (i.e., more than four different sequences) [2].

In this review, we cover current hot topics on the modification of PLS and quenchers, as well as recent applications that depart from those of classic MBs.

Modification of the photoluminescent species

MBs make use of PLS as reporter groups. Improving MB design, therefore, involves finding new and improved PLS. In this section we review some of the latest advances in this area.

Metal complexes

One of the most studied metal complexes is tris(2,2’-bipyridine)ruthenium(II), (Ru(bpy)3)2+, which was first applied by Wilson and Johansson [17] as a reporter group for MB design (Fig. 2a). A Black Hole Quencher-2 (BHQ-2) was attached to the 5’ end of the oligonucleotide, and the (Ru(bpy)3)2+ was attached to the 3’ end. The recognition of the complementary DNA sequence was detected by photoluminescence and electrochemiluminescence. Interestingly, it was found that the signals derived from electrochemiluminescence were free from background photoluminescence and scattering.

Fig. 2
figure 2

Metal complexes as PLS in MB composites: a MB containing a ruthenium complex as a PLS and an organic quencher [17]; b MB containing a ruthenium complex as a PLS on the surface of a gold electrode [18]; c MB containing metallophthalocyanines at both sides of the strand [19]; d MB containing a lanthanide complex as a PLS [20]; e MB containing a europium complex as a PLS [21]

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Zhang et al. [18] developed another ruthenium-complex-based electrochemiluminescent MB probe (Fig. 2b). In this approach, a thiolated hairpin DNA tagged with (Ru(bpy)3)2+ was self-assembled on the surface of a gold electrode. In the absence of target DNA, the hairpin DNA is in the folded configuration and its termini are held close to the surface of the electrode, allowing the generation of a strong electrochemiluminescence signal. Upon hybridization with target DNA, the double helix is formed and removes the ruthenium complex tag from the electrode surface. Thus, a decrease of the electrochemiluminescence signal can be detected. Such an “on–off” mechanism was applied to detect single-stranded DNA.

Water-soluble metallophthalocyanines are one class of metal complexes that were also introduced into the design of MBs by Nesterova et al. [19]. They are near-IR PLS with unique photophysical characteristics, such as (1) an intrinsic propensity to form nonfluorescent H-type dimers and (2) diminished metallophthalocyanine dimerization when the MB is hybridized to a complementary target. Figure 2c shows the basic design of the metallophthalocyanine-dimer-based MB. Before hybridization of the MB to the target, two identical metallophthalocyanine molecules form a nonphotoluminescent H-type dimer. Upon hybridization, the loop opens, disrupting the metallophthalocyanine dimer and restoring photoluminescence.

Krasnoperov et al. [20] reported a series MB probes based on lanthanide complexes of Tb3+, Eu3+, Sm3+, and Dy3+. Because of their low absorbance (ε < 1 M-1 cm-1), lanthanides must be “photosensitized,” which is achieved by tethering the lanthanide ion (through chelation) to an appropriate organic dye. Diethylenetriaminepentaacetic acid derivatives are used as chelating agents to attach the lanthanide ions. They are first coupled with oligonucleotides, followed by the addition of a lanthanide metal cation for chelation (Fig. 2d). Time-resolved gating was applied to reduce the interference from background emission, and detection limits can therefore be as low as 1 pM.

Li et al. [21] designed an MB with a Eu3+ complex with chlorosulfonylated tetradentate β-diketone as polydentate ligand and a BHQ-2 quencher (Fig. 2e). In their design, instead of chelating initially the Eu3+ cations with β-diketone derivatives, 1.0 equiv of Eu3+ ions was added after the hybridization of the metal-free MB with the target DNA in buffer solution. The photoluminescence response increased with increasing Eu3+ concentration. The long-lived luminescence of Eu3+ is well distinguished from the short-lived background fluorescence and scattered light by using time-resolved methods. This MB probe has been used to qualitatively and quantitatively detect a target DNA sequence in cell growth media.

Quantum dots

Compared with organic dyes, photoluminescent semiconductor nanocrystals, such as quantum dots (QDs) made of CdSe and CdTe cores, have several unique properties, which promise to provide a significant advantage in bioanalytical and sensing applications [22]. Some of these advantages include high quantum yields, narrow, symmetric, and stable photoluminescence, and size-dependent and tunable adsorption and emission [23], which make them attractive as PLS in MB probes. One of the most used QDs in the design of MBs consists of a CdSe core material coated with a ZnS shell [24]. The shell passivates the core, protects it from oxidation and bleaching, and at the same time significantly improves the photoluminescence [25–28].

ZnS-capped CdSe QDs were chosen to replace conventional organic dyes by Kim et al. [29]. The CdSe–ZnS core–shell QDs (3.7 nm in diameter) were first modified with mercaptoacetic acid and then conjugated with a 25-base oligonucleotide, which had the quencher DABCYL at the 3’ end (Fig. 3a). The increase in photoluminescence was enough for target DNA monitoring using fluorescence resonance energy transfer (FRET) of the QD–DABCYL couple, even though its calculated FRET efficiency is less than that of the conventional 6-FAM/DABCYL donor–acceptor pair. The same group [30] later developed multicolor QD-based hybrid MB probes with emissions at 525, 565, and 605 nm. The optical and gel-electrophoretic characterization revealed the target DNA detection limits to be 8 ng. A similar reasoning was applied by Wu et al. [31] to synthesize MBs for direct photoluminescence in situ hybridization of β-lactamase genes in the bacterium Escherichia coli.

Fig. 3
figure 3

MB architectures using quantum dots (QDs) as PLS: a MB containing a QD as a PLS and an organic quencher [29–32, 40]; b MB containing a QD as a PLS and a gold nanoparticle as a quencher [33]; c MB containing a QD and an organic dye to form a dual-emission MB [34]; d MB containing a QD with a silica shell and an organic dye for a more stable dual-emission MB [35]; e MB containing a QD as a PLS and graphene oxide (GO) as a quencher [23]

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Chen et al. [32] discovered that QD-based MBs have the property of being retained in the cytoplasmic compartment of cells and thus can be used to measure endogenous gene expression. Specifically, the expression of c-myc in MCF-t breast cancer cells was measured by quantifying the total photoluminescence signal emanating from individual cells.

With improved quenchers, Yeh et al. [33] described a hybrid photoluminescent nanoprobe composed of a nuclease-resistant MB backbone, a CdSe–ZnS core–shell QD as the donor, and a gold nanoparticle (AuNP) as the quencher (Fig. 3b). By using an AuNP to QD ratio of 6:1, they achieved a 7.3-fold increase in fluorescence signal upon target binding. In living cell experiments, a hexahistidine-appended Tat peptide was self-assembled onto the QD surface to provide nearly 100% noninvasive delivery of the MB within 2 h. By direct visualization of the photoluminescence obtained from the probe when it was bound to the newly synthesized CVB6 virus RNA, this AuNP-based MB probe provided sensitive and real-time detection of infectious viruses as well as real-time visualization of cell-to-cell virus spreading.

Recently, Liu et al. [34] reported a two-photon-excitation MB using a water-soluble CdSe–ZnS core–shell QD as the energy donor and 6-carboxy-X-rhodamine as the acceptor (Fig. 3c). This MB probe was constructed via specific binding between streptavidin on the surface of QDs and biotin tagged to the hairpin structure. This QD-based two-photon-excited MB can distinguish complementary, noncomplementary, and single-base-mismatch DNAs with high specificity in aqueous buffer. Additionally, two-photon-excitation MBs were used to discriminate target DNA from autofluorescence in a strongly fluorescent medium.

Wu at al. [35] coated CdSe–ZnS core–shell QDs with a silica shell and covalently coupled them to MBs (Fig. 3d), since traditional water-soluble QDs coupled with MBs suffer from problems such as photoluminescence degradation, and chemical and colloidal instabilities in harsh environments (e.g., low-pH buffer). The QDs had a high quantum yield over a wide range of pH values from 1 to 14. The small diameter (less than 10 nm) of those QDs allows efficient energy transfer in MBs. The reported QD-based MB probes are capable of detecting target DNA at 0.1 nM concentration within 15 min and are capable of differentiating the target DNA from single-base-mismatched DNA.

In addition to CdSe, QDs with a CdTe core have been extensively applied in MB design and bioanalysis [36–39]. Dong et al. [23] designed a novel MB based on FRET from CdTe QDs to graphene oxide (GO) (Fig. 3e). The CdTe QDs were capped with mercaptoacetic acid and coupled with oligonucleotides in buffer solution with N-ethyl-N-(dimethylaminopropyl)carbodiimide. The MB detection method was based on GO binding to the QD-based MB. After addition of GO (0.1 μg/mL) to the solution of QD-based MBs (150 nM), the photoluminescence intensity of the QD-based MBs reduced rapidly to 10% of the original intensity, indicating the strong quenching effect of the GO on the photoluminescence of QDs. However, when the QD-based MB was hybridized to target DNA, binding of GO was diminished and the photoluminescence was not efficiently quenched. The threshold of detection was 12 nM.

A novel method that combines capillary electrophoresis with MBs based on CdTe QDs for simultaneous detection of dual base mutation was recently reported by Li et al. [40]. Capillary electrophoresis is a highly efficient separation technique, and has been used in the hybridization analysis of MBs with DNA targets [41, 42], in which an effective separation of the MB–target hybrid from the unhybridized MB can be achieved. The separation of the MB–target hybrid avoids the interference from the signal from the unhybridized MB, eliminating false-positive results completely. In their work, two MB probes were designed by conjugating CdTe QDs with emission at 585 and 650 nm to different DNA oligonucleotide sequences. The hybridization of QD-based MBs with different DNA targets was monitored by capillary electrophoresis, showing that QD-based MB probes specifically hybridize only with their complementary DNA sequences. Target DNA identification was observed to have a high sensitivity of 16.2 pg. Furthermore, the simultaneous detection of dual single-base mutations in a given DNA oligonucleotide can be achieved by capillary electrophoresis using the two aforementioned QD-based MB probes.

Pyrene excimer

Pyrene, because of its tendency to form excimers, has been used as another kind of PLS, the emission of which can be regulated through the formation of π–π stacking aggregates. When an excited-state molecule is brought close to a molecule in the ground state, an excimer is formed whose emission is redshifted from the emission of the monomer [43]. To date, pyrene has been employed as a PLS to signal the presence of small molecules [44], nucleic acids [45], and proteins [46].

Fujimoto et al. [47] developed a class of MBs with pyrene monomers at both the 5’ end and the 3’ end (Fig. 4a). Upon addition of a target sequence (0.1–1.0 equiv), the color of the MB solution turned from yellow-green (λ em = 498 nm) owing to the pyrene excimer to pale blue (λ em = 382 nm) owing to monomeric pyrene. The spectral changes could be followed up at a low concentration of 1 nM MB with a standard fluorescence spectrometer. One equivalent of target completed the switching of the emission and excess target led to no spectral change.

Fig. 4
figure 4

MBs based on excimer photoluminescence: a MB containing two pyrene (P) molecules at opposite sides of the strand [43, 47]; b MB containing two pyrene (P) molecules at the same sides of the strand without quenchers [48]; c MB containing two pyrene (P) molecules at the same sides of the strand with quenchers [49]; d MB containing two pyrene (P) molecules at opposite sides of the strand with extra length of the sticky end at one side [53]; e MB containing one pyrene (P) molecule incorporated into the middle region of the stem [54]; f MB containing two pyrene (P) molecules incorporated into the middle region of the stem [55]; g MB containing two nonnucleosidic pyrenes (Y) and perylenediimides (E) as building blocks in the stem [11, 56]; h MB containing perylene-3,4,9,10-tetracarboxylic acid bisimides (P) intercalated into the stem [57]

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Chen et al. [43] made use of dual-pyrene-labeled MBs to detect ribonuclease RNase H. The pyrene moieties were brought together by the beacon’s hairpin structure, exhibiting excimer emission at 485 nm. Binding of the DNA beacon to RNA opens up the hairpin structure and thus spatially separates the pyrene moieties. The RNA–DNA hybrid, which serves as the substrate for the RNase H cleavage, has a low photoluminescence background at 485 nm. After the addition of the enzyme, only the RNA strand will be cleaved from the duplex, which sets free the MB. The absence of target leads to the restoration of the hairpin structure, bringing the pyrene moieties back together and producing a dramatic photoluminescence enhancement at the excimer emission (485 nm).

Yamana et al. [48] reported a bispyrene-labeled MB where two pyrene fluorophores are attached at the 5’ end of the oligonucleotide probe and the 3’ end is free (Fig. 4b). The MB actually acts as a probe carrier instead of recognizing the target DNA directly. In this assay, an MB first hybridizes with an exchangeable strand that can specifically hybridize with the target oligonucleotide, providing a partially double stranded duplex. The strand-exchange reactions between this partially double stranded probe and the single-stranded target DNA were monitored by changes in photoluminescence from the monomer to the excimer. The presence of a mismatched base in single-stranded DNA can be detected as well on the basis of the different reaction rates for strand exchange from fully matched DNA.

Conlon et al. [49] reported multiple-pyrene-labeled MBs, in which two pyrene monomers are linked symmetrically to the 5’ end and a single DABCYL molecule is linked to the 3’ end of a MB sequence (Fig. 4c). The conjugation of additional monomers at the same terminal (5’ end) magnifies the photoluminescence intensity of the excimers. When the MBs were titrated with 1 equiv of target DNA, the maximum intensity increase of the excimer emission was achieved. The relatively long fluorescence lifetime (40 ns) and the large Stokes shift (130 nm) were utilized for time-resolved fluorescence measurement and background discrimination. Later, this group developed a DNA-amplified detection method based on the combination of the hybridization chain reaction [50–52] and pyrene-labeled MBs [53]. In the absence of target DNA, both MB probes are in the closed form, and the two pyrene moieties are spatially separated by the extra length of the sticky end (Fig. 4d). Therefore, only the monomer emission is observed. Upon addition of target DNA, a hybridization chain reaction occurs, whereby the 3’-end sequence of one MB probe was placed close to the 5’-end sequence of another MB probe, accompanied by the enhancement of excimer emission.

Saito et al. designed end-free MBs by incorporating pyrene-modified pyrrolocytidine (pC) [54] and 2’-deoxyguanosine (dG) [55], respectively, into the middle region of MB stems. The “off” hairpin state originates from the quenching of the pyrene by the opposite dG by forming a pC–G base pair (Fig. 4e) or by the opposite C of the dG–C base pair (Fig. 4f). In the pC–G model, MBs were designed in which pyrene-labeled pC was placed three bases apart from the 3’ terminus. The researchers found that the capability of detecting target DNA is not affected by the stem sequence and the location of pC in the stem. In the dG–C model, the design involves an emissive excimer MB containing two pyrene fluorophores separated by two bases in the middle region of the stem. In the closed form, the two pyrene molecules cannot sterically interact with each other and excimer formation is precluded. In the open form, the two pyrene groups located in a single-stranded region can interact with each other, resulting in the formation of an excimer. Therefore, the MB shows only monomer emission in its closed form, whereas in the presence of target DNA it exhibits excimer photoluminescence.

In a different approach, Häner et al. [11, 56] intercalated nonnucleosidic pyrenes and perylenediimides as building blocks into the stem of MBs (Fig. 4g). The directed assembly of chromophores in the stem of MBs was used as an on–off switch. In the native hairpin structure, excimer formation between pyrene derivatives was prevented by the formation of donor–acceptor complex with perylenediimide. Upon hybridization with the target, perylenediimide and pyrene units were separated, allowing the formation of an excimer. The researchers also showed that the reduction of the stem length from four to two base pairs improved base-mismatch discrimination. In a similar approach, Menacher and Wagenkenecht [57] reported an MB design where pyrenes are replaced with perylene-3,4,9,10-tetracarboxylic acid bisimide chromophores (Fig. 4h).

Modification of the quencher

An intense photoluminescence background is generated by incomplete quenching of the PLS in the MB, which greatly decreases the signal-to-background ratio and thus, the selectivity of the assay [58]. In the following section we review some of the recent efforts to produce novel and improved quenchers.

Nanoquencher

Intramolecular quenchers (nanoparticles and nanowires)

Tuning the photophysical properties of nanosized materials can be achieved by changing their shape, size, and composition; therefore, their quenching properties can be optimized to match a specific PLS. Particularly, AuNPs have attracted considerable interest because of their unique optical characteristics. [59, 60] The unusual properties of DNA-modified AuNPs, including the ability to enter cells, effective protection of DNA from enzymatic degradation, and an extraordinary intracellular stability, make them attractive for intracellular molecular analysis and other bioapplications [60].

Dubertret et al. [16] first applied 1.4-nm-diameter AuNPs as a quencher for MBs and achieved 100 times better quenching efficiency in the near-IR region in comparison with the first reported MB in 1996. The 5’ end of the oligonucleotide was covalently attached to the AuNP by coupling free sulfhydryl at the end of the oligonucleotide with N-propylmaleimide on the surface of the nanoparticles (Fig. 5a). With four different kinds of organic dyes, the average quenching efficiency reached up to 99.45%, and single-base mutation could thus be discriminated.

Fig. 5
figure 5

Intramolecular and intermolecular nanoquenchers in MBs. a MB containing a gold nanoparticle as a quencher [16, 33, 58, 60, 65, 66]; b MB containing a gold nanoparticle as a quencher (no stem) [61]; c MB containing a silver nanoparticle as a quencher [67]; d MBs containing a nanobarcode particle as a quencher [68–73]; e MB containing a single-walled carbon nanotube as a quencher [77]; f MB containing an organic dye as a PLS and GOs as a quencher [76, 78, 80]; g MB containing a QD as a PLS and GO as a quencher [23, 79]

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Maxwell et al. [61] developed an AuNP-based “molecular beacon” (MB) without a stem structure. In their design, a 2.5-nm AuNP functions as both a nanoscaffold and a nanoquencher (Fig. 5b). Surface-enhanced Raman scattering studies showed that the PLS can be adsorbed reversibly on the surface of these colloidal nanoparticles [62, 63] and their photoluminescence could thus be quenched. Upon target binding, the constrained conformation is opened and the PLS is separated from the particle surface. The detection mechanism is similar to that of a MB.

Shan et al. [64] described a DNA electrochemiluminescence sensor with a CdS:Mn nanocrystal film as the PLS and 5-nm AuNPs as quenchers. The combination of CdS:Mn nanocrystals and AuNPs in a MB scaffold allows selective target detection. As described in “Modification of the photoluminescent species,” Yeh et al. [33] designed a hybrid photoluminescent probe based on CdSe–ZnS core–shell QDs as donors and AuNPs as quenchers (Fig. 3b), and which was exploited for sensitive and real-time detection of infectious viruses as well as real-time visualization of cell-to-cell virus spreading.

Owing to the excellent quenching properties and biocompatibility of AuNP-based MBs, their bioapplication has been further broadened. Qiao et al. [60] attached two different MBs to a 20-nm AuNP, which allowed the simultaneous detection of two types of tumor mRNAs in breast cancers by means of fluorescence imaging [60]. The detection limit of these MBs reached 0.3 nM. They also used AuNP-based MBs as a tumor-mRNA-dependent drug carrier for controlled release of doxorubicin and intracellular imaging [65]. In this case, doxorubicin was physically intercalated into the PLS-free MBs, which specifically targeted intracellular cyclin D1 mRNA (a tumor mRNA of breast cancer). The AuNP-based MBs can carry doxorubicin, quench its photoluminescence, and decrease its cytotoxicity. When the MB is selectively bound to cyclin D1 mRNA, doxorubicin is released effectively and induces apoptosis. The photoluminescence intensity of doxorubicin increases upon increasing the target concentration. Another application of AuNP-based MBs is as lateral flow strip biosensors; these were developed by Mao et al. [58] and He at al. [66] for rapid detection of nucleic acid sequences with high sensitivity and low cost.

In addition to AuNPs, silver nanoparticles were employed by Peng et al. [67] as nanoquenchers. They assembled MBs with tetramethylrhodamine (3’ end) attached to the silver nanoparticles via a 5’-thiol (Fig. 5c). With different surface roughness, particle sizes, and amounts of silver on the surface, the detection performance differed significantly. Their reported limit of detection was approximately 100 pM.

Metal (gold, silver) nanowires, also known as nanobarcode (NBC) particles, are another class of nanoparticles that can be utilized as MBs. They are normally fabricated by electroplating an inert metal (gold, silver) sequentially into alumina templates, followed by dissolving the template. Keating’s group [68–72] has studied in depth the use of gold and silver nanowires as quenchers for MBs (Fig. 5d). The NBC particles in their studies were generally 250 nm × 6 μm, and contained six metallic segments.

NBC particles have an advantage over other MB designs in that they can readily detect multiple targets with a single assay. Sha et al. [68] demonstrated the simultaneous detection of five pathogens, hepatitis A virus, hepatitis C virus, West Nile virus, human immunodeficiency virus, and severe acute respiratory syndrome virus from a multiplexed real-time PCR using a single NBC particle.

After the initial proof-of-concept experiments, use of NBC particles was further explored by investigating the effect of the label position and the secondary structure in oligonucleotide probes as a function of the hybridization buffer. Hybridization buffers, such as 0.3 M phosphate-buffered, affect the percentage of double-stranded probes on the surface of the NBC particles after exposure to complementary DNA. The reported metal–dye separation dependence for unstructured surface-bound oligonucleotides is highly sensitive to the hybridization efficiency, owing to substantial changes in DNA extension from the surface upon hybridization. Differences in the photoluminescence patterns on gold and silver were utilized in their assays as a function of not only chromophore identity but also metal–dye separation [69]. Optimized MB probes had K d of 1.7 nM, with a detection limit of 100 pM, yet demonstrated high selectivity even after storage for 3 months. These assays were limited though by incomplete quenching [70].

Later, this group observed that the coverage of MBs can be controlled by varying the MB concentration, by varying the buffer ionic strength, and by addition of short hydroxy-terminated alkanethiol diluent molecules during probe assembly onto the NBC particle surface. The optimal coverage for the detection of a target sequence is about 1012 molecules per square centimeter. For both probes the surface coverage and sensor performance differ for different probe sequences [71]. The hybridization efficiency is also dependent on the probe surface coverage, reaching a maximum of 90% at intermediate coverages of 1.13 × 1012–2.0 × 1012 probes per square centimeter and dropping to 20% or less at higher or lower coverages. Additional nucleotides on the 3’ end of the complementary target sequence (i.e., the end near the nanowire surface) had a much greater impact on the hybridization efficiency as compared with nucleotides added to the 5’ end [72].

Sha et al. [73] also assembled dual-mode MBs on metal NBC particles with both surface-enhanced Raman scattering and a photoluminescence signal. In their study, similar NBC particles 300 nm wide and 6-9 μm long were used as the quencher and scaffold. A hepatitis C virus reverse transcriptase PCR product was detected using this dual-mode beacon.

Intermolecular quencher (graphene oxide and nanotubes)

Both single-walled carbon nanotubes (SWNTs) and GO have been utilized as intermolecular quenchers. It has been demonstrated that single-stranded DNA can interact noncovalently with SWNTs [74, 75]. Double-stranded DNA can also interact with SWNTs, but its affinity is much weaker than that of single-stranded DNA. The difference in the binding interaction of SWNTs with single-stranded DNA and double-stranded DNA provides the basis for their use in the construction of MBs. In the assay, SWNTs and GO serve as both a “nanoscaffold” for the oligonucleotide and a “nanoquencher” for the PLS [76].

SWNTs are a novel class of universal quenchers which have been used in the construction of MBs [77]. The efficient absorption of hairpin-structured oligonucleotides to the surface of SWNTs or GO is likely due to hydrophobic and π-stacking interactions between the nucleobases and SWNTs or GO [78]. In the approach of Yang et al. [77] (Fig. 5e), a self-assembled complex of photoluminescent single-stranded DNA and SWNTs works as an efficient MB for detecting single-nucleotide variations in DNA. Upon hybridization with the target, competitive binding of the target over the carbon nanotubes produces the photoluminescence signal enhancement. During the experiment, more than 98% quenching was observed for an MB concentration from 50 to 200 nM. The nanotube surface with an adsorbed single-stranded DNA strand is much less dependent on temperature than a conventional MB; therefore, the SWNT-based MBs function well at both room temperature and at relatively high temperatures. This MB has a signal-to-background ratio of about 15, with a selectivity coefficient of 0.472.Footnote 2

In a different approach, Lu et al. [78] developed GO-quenched MBs with either fluorescein or Cy5 at the 3’ end (Fig. 5f). Owing to hydrophobic and stacking interactions between hairpin-structured oligonucleotide and GO, up to 99.3% quenching of the photoluminescence in the absence of a complementary target DNA was observed. In the presence of a target DNA, the formation of the duplex structure disturbs the interaction between the oligonucleotide and GO, and releases the oligonucleotide from GO, resulting in restoration of photoluminescence of the PLS. The strong quenching efficiency allows a detection limit of 2 nM. The selectivity coefficient reached 0.246 for the GO–FAM-based MB and 0.267 for the GO–Cy5-based MB for a perfectly matched target over a single-base-mismatched target.

Dong et al. [23] also reported GO-quenched MBs based on FRET between QDs and GO (introduced in “Modification of the photoluminescent species” as a QD fluorophore) (Fig. 5g). Upon target recognition by the MB, an increase in the QD–GO distance, facilitated by a weakened DNA–GO interaction, significantly hindered FRET, producing an increase in the photoluminescence of the QDs. In their experiment, 0.1 μg/mL GO was selected for analytical purposes. Yi et al. [79] used a 1,000 times concentrated GO solution (0.1 mg/mL) for quenching similar quencher-free MBs and obtained a photoluminescence increase in the signal-to-background ratio of 31.0.

In the last few years, applications of GO-based MBs in bioanalysis have been gradually broadening. Zhang et al. [76] labeled GO-based MBs with the dyes fluorescein isothiocyanate, Cy3, and Cy5 for multiplex DNA detection. The limit of detection of target DNA was approximately 1 nM. Wu et al. [80] presented a GO-based MB for DNA phosphorylation detection without DNA amplification. Polynucleotide kinase was detected at a level as low as 0.001 units per milliliter from the derived calibration curve.

Metal surfaces

Immobilization has been found to be an effective way to improve the hybridization of linear DNA probes. Du et al. [81] and Strohsahl et al. [82] utilized a metal surface as a functional part of the MB and other MBs had their quencher molecules at the end of the DNA strand, with the material surface only serving a passive role.

Du et al. [81, 83] used the principle of the MB in solution but translated it to a planar gold surface, where the metal surface acts as a quencher and organic dyes work as PLS. MBs were attached to the surface by immersing the substrate in an MB–mercaptopropanol solution at a ratio of 1:10, followed by rinsing the substrate with hot water. Long incubation periods and a relatively low concentration of mercaptopropanol provided gold surfaces with a large amount of bound MBs. However, some interstitial space between probes is required for high hybridization efficiency, since the hybridization efficiency is determined by both the probe secondary structure and the surface distribution on the substrate. In their design, portions of Staphlococcus aureus FemA and mecR methicillin-resistance genes were incorporated into the construction of the loop portion of MBs. Strohsahl et al. [84] extended the application of the gold-immobilized MBs by utilizing partial gene folding for identification of a putative sequence. The partial gene folding is a portion of the pag gene of Bacillus anthracis, which is the causative agent of anthrax [85]. Later, the same group replaced organic dyes with CdSe nanocrystals for the production of gold-immobilized MBs [86].

Fan et al. [87] reported another kind of immobilized MBs based on electrochemical detection. In the absence of target DNA, the hairpin structure holds ferrocene at the 5’ end close to the electrode surface, and thus produces an intense signal. The hybridization of target DNA decreased the electrochemical signal by moving the ferrocene group away from the electrode surface. The limit of detection reached 10 pM by cyclic voltammetry.

Luan et al. [88] immobilized MBs on gold films. Then they installed the modified film in a flow cell for experiments and obtained a detection limit of 0.5 fM. Wu et al. [89] developed a transducer that combines the use of MB and Escherichia coli DNA ligase for electrochemical detection of target DNA. Other applications of gold-surface-immobilized MBs include the detection of thrombin [90] and adenosine 5’-triphosphate [91].

Other quenchers

In addition to the two main types of novel quenchers discussed, some other efficient quenchers have been studied, such as metal complexes [92] and superquenchers [93].

Brunner and Kraemer [92] introduced Cu2+ complexes as new intramolecular quenchers in MBs. In their case, the intramolecular interaction of fluorescein and a Cu2+ complex is supported by the formation of the hairpin structure (Fig. 6a). The photoluminescence of fluorescein is restored upon addition of fully complementary 25mer single-stranded target DNA since intramolecular interaction of fluorescein with the Cu2+ complex becomes sterically impossible in the rigid, linear DNA duplex. According to the authors, the quenching process is triggered by intramolecular coordination of a phenolate donor of fluorescein to a free coordination site of the copper(II) 5-(2-pyridinyl)pyrazole (pypz) complex, resulting in the formation of a nonphotoluminescent Cu(fluorescein)(pypz) complex.

Fig. 6
figure 6

a Copper complex [92] and b superquenchers in MB architectures [93]

Full size image

Yang et al. [93] assembled an array of quencher molecules to produce superquenchers for use in MBs. In their study, three DABCYL molecules were assembled together to the 5’ end of the MBs (Fig. 6b). The superquencher MB exhibited a quenching efficiency as high as 99.5% and a photoluminescence increase of 320-fold after hybridization, which is a significant improvement compared with the 14-fold signal-to-background ratio from the original MB with only one DABCYL molecule.

Other applications

Owing to their high specificity, selectivity, and sensitivity, MBs have the potential of being a general platform for sensing molecules other than oligonucleotides, as well as for the production of singlet oxygen, and as molecular carriers. Here, we describe some recent nonconventional applications of MBs.

Detection of ions and molecules

For detection of metal ions, MBs are generally hybridized with an aptamer, which acts as an ion recognition element. Binding with ions results in a disruption in the hybridization between the MB and the aptamer, which provides the signal change. Shi et al. [94] reported an aptamer-based MB for potassium ion detection. A MB that is partially complementary with an aptamer was labeled with pyrene moieties at both ends. In the presence of K+, the MB was displaced from the aptamer, forming a hairpin structure, which was accompanied by an increase of the excimer photoluminescence of pyrene. However, it provided only monomer emission in the absence of K+. The photoluminescence intensity of the pyrene excimer is proportional to the concentration of K+ in the range from 0.6 mM to 20 mM, and a detection limit of 0.4 mM was achieved. The combination of an aptamer and an MB was also applied for the selective sensing of lysozyme by Tang et al. [95]. In their design, the MB can bind to E. coli SSB protein and produce an increase in photoluminescence. In the absence of lysozyme, the SSB protein is wrapped by the aptamer, which blocked its binding to the MB. However, the lysozyme has a stronger affinity for the aptamer and its presence makes the free SSB protein interact with the MB. The increase in photoluminescence signal is proportional to the amount of lysozyme.

Following the discovery by Ono and Togashi [96] that Hg2+ can specifically interact with thymine bases to form thymine–thymine base pairs (T–Hg2+–T), this metal-dependent pair of two nucleobases has been widely utilized for optical sensors [97, 98]. Xu et al. [99] reported a MB-based Hg2+ sensor which integrates the high selectivity of T–Hg2+–T coordination and the sensitivity of a MB framework. The presence of Hg2+ helps the hybridization of the loop portion of the MB with the thymine–thymine mismatched target DNA and produces the enhancement of the photoluminescence.

A similar design of thymine–thymine base pairs in the MB stem was applied for the detection of glutathione (GSH) and cysteine (Cys) [100]. The approach is based on the competitive ligation of Hg2+ ions by GSH/Cys and thymine–thymine mismatched in the self-hybridizing stem of the MB. The extraction of Hg2+ ions by GSH/Cys destabilizes the MB stem and results in switching the MB to the “on” state.

Zhang et al. [101] developed DNAzyme catalytic beacons with a MB for detection of Pb2+. The DNAzyme substrate strand binds with the loop of the MB to form a hybrid structure. Addition of metal ions cleaves the substrate, cutting the MB into two pieces, resulting in a separation from the quencher and producing an enhancement of photoluminescence. By replacement of DNAzyme with aptamers, the modified MBs can be extended to sense other biomolecules such as adenosine [97]. Wang et al. [102] developed a solid-state MB using a gold surface as a quencher. This system was combined with a polydimethylsiloxane microfluidic channel to detect a single-stranded DNA binding protein. Such proteins are abundant and essential in cells: they interact with DNA to organize its packing, to regulate transcription, and to perform replication and repair. Transcription factors are one subset of these proteins. Similarly to the target oligonucleotide, the single-stranded DNA binding protein binds with the MB, making a rigid rodlike structure, which results in the recovery of its photoluminescence.

Control of singlet oxygen production for photodynamic therapy

Chen et al. [103] reported a photodynamic MB (PMB) with tumor-specific mRNA-triggered control of singlet oxygen (1O2) generation. The mRNA-PMB consists of a standard MB architecture where a photosensitizer is attached at one end of the strand and a quencher is attached at the opposite end, such that 1O2 production is inhibited until hybridization of the antisense oligonucleotide with tumor-specific mRNA. The 1O2 near-IR photoluminescence was reduced by 93% when compared with that for a PMB without a quencher. When the target was added, there was a 63% restoration of 1O2 photoluminescence when compared with that for PMB without a quencher. The MB displayed efficient cellular uptake without the aid of transfection agents, owing to the existence of a pyropheophorbide component. Later, the same group made use of a flexible solid-phase approach for completely automated synthesis of PMBs, where a linear superquencher was positioned at the 5’ end [104]. The high quenching efficiency of linear superquenchers displays near 90-fold restoration upon activation.

Control of hybridization

To achieve the regulation of hybridization of MBs with target DNA, Yang et al. [105] employed T–Hg2+–T to replace the Watson–Crick duplex at the stems of MBs. Because Hg2+ can stabilize T–Hg2+–T base pairs, the desirable kinetic response and selectivity of MBs at different temperatures can be controlled by changing the Hg2+ concentration. Later, Wang et al. [106] constructed light-activatable caged MBs . In their approach, the MBs are caged by locking two stems with a photolabile biomolecular interaction or covalent bond. Biotin–avidin interaction or a triazole linkage are applied to lock the stems of the MBs via a photocleavable linker bearing an o-nitrobenzyl moiety, which is extensively used as a caging agent owing to its fast photolysis kinetics and efficient uncaging capabilities. The hybridization activity of MBs with target DNA can thus be regulated by light.

Concluding remarks

Since the first MB was reported, conventional MBs have been widely modified to improve their selectivity, sensitivity, and specificity. Different kinds of PLS, including metal complexes, QDs, and pyrenes, have been developed to function as reporter groups, and nanoquenchers have been utilized as a main class of high-efficiency quenchers. In addition, MBs have been utilized as a platform for other nonconventional applications, including detection of metal ions and small molecules, production of molecules (singlet oxygen), and control of DNA hybridization. However, even with all these recent advances in MB design, there is still room for improvement. PLS with larger extinction coefficients, better quantum yield, and longer photoluminescence lifetimes are required to make more sensitive sensors. Also, false-positive signals, either by incomplete quenching or by nonspecific opening of the MB, are a real limitation, especially for their use in cellular environments. New ways to improve specificity, reduce nonspecific opening, or discriminate nonspecific from specific binding in vivo are needed. It is expected that new techniques will start to find application in DNA and mRNA detection both in vitro and in vivo. For example, hybridization chain reaction has been recently used for cellular mRNA tracking [107]. Techniques that allow better resolution by reducing the cellular autofluorescence background are also of importance for in vivo detection of oligonucleotides. For example, two-photon excitation is a promising technique for autofluorescence discrimination, since the excitation can be chosen to minimize the absorption cross section of undesirable cellular dyes. A potentially useful photophysical phenomenon that can be used for the same purpose is photoluminescence upconversion. In photoluminescence upconversion, the excitation light has a longer wavelength than the emission, a process often called anti-Stokes shift. Furthermore, it is likely that processes such as chemiluminescence and techniques such as fluorescence lifetime imaging microscopy, which are nonconventionally used in combination with MBs, will be used to address these and new challenges in DNA and RNA detection.