Biosensors and Bioelectronics 59 (2014) 64–74

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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Optical aptasensors for quantitative detection of small biomolecules: A review Chunjing Feng, Shuang Dai, Lei Wang n Key Laboratory of Natural Products Chemical Biology, Ministry of Education, School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 18 December 2013 Received in revised form 27 February 2014 Accepted 6 March 2014 Available online 15 March 2014

Aptasensors are aptamer-based biosensors with excellent recognition capability towards a wide range of targets. Specially, there have been ever-growing interests in the development of aptasensors for the detection of small molecules. This phenomenon is contributed to two reasons. On one hand, small biomolecules play an important role in living organisms with many kinds of biological function, such as antiarrhythmic effect and vasodilator activity of adenosine. On the other hand, the concentration of small molecules can be an indicator for disease diagnosis, for example, the concentration of ATP is closely associated with cell injury and cell viability. As a potential analysis tool in the construction of aptasensors, optical analysis has attracted much more interest of researchers due to its high sensitivity, quick response and simple operation. Besides, it promises the promotion of aptasensors in performance toward a new level. Review the development of optical aptasensors for small biomolecules will give readers an overall understanding of its progress and provide some theoretical guidelines for its future development. Hence, we give a mini-review on the advance of optical aptasensors for small biomolecules. This review focuses on recent achievements in the design of various optical aptasensors for small biomolecules, containing fluorescence aptasensors, colorimetric aptasensors, chemiluminescence aptasensors and other optical aptasensors. & 2014 Elsevier B.V. All rights reserved.

Keywords: Biosensors Aptasensors Aptamers Optical analytical techniques Small biomolecules

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Fluorescence aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 2.1. Labeled fluorescence aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 2.1.1. Typical FRET-based fluorescence aptasensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 2.1.2. Nanoparticle-based fluorescence aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 2.1.3. Other labeled fluorescence aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 2.2. Label-free fluorescence aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 2.2.1. DNA intercalators-based fluorescence aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 2.2.2. Abasic-site-binding dyes-based fluorescence aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 2.2.3. Metal nanomaterials-based fluorescence aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 2.2.4. Other label-free fluorescence aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Colorimetric aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 3.1. Au nanoparticles aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 3.1.1. DNA-functioned AuNPs aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 3.1.2. Label-free AuNPs aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Abbreviations: EIAs, enzyme immunoassays; ELISAs, enzyme-linked immunosorbent assays; FRET, fluorescence resonance energy transfer; MB, molecular beacon; PNK, polynucleotide kinase; CAMB, catalytic and molecular beacon; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; EB, ethidium bromide; AP site, apurinic/ apyrimidinic site; ATMND, 2-amino-56,7-trimethyl-1,8-naphthyridine; AgNCs, Ag nanoclusters; CuNPs, Cu nanoparticles; MG, Malachite green; AuNPs, Gold nanoparticles; NMM, N-methyl mesoporphyrin IX; SPR, surface plasmon resonance; HRP-mimicking DNAzyme, horseradish peroxidase-mimicking DNAzyme; ABTS, 2,2-azino-bis(3ethylbenzothiazoline-6-sulfonicacid) diammonium salt; TMB, 3,3,5,5-tetramethylbenzidine sulfate; CL, chemiluminescence; TMPG, 3,4,5-trimethoxyl-phenylglyoxal; CRET, chemiluminescence resonance energy transfer; ICIA, indirect competitive inhibition assay; AuNRs, gold nanorods; SERS, surface-enhanced Raman scattering; RS, resonance scattering; DLS, dynamic light scattering n Corresponding author. Tel.: þ 86 531 88363888. E-mail address: [email protected] (L. Wang). http://dx.doi.org/10.1016/j.bios.2014.03.014 0956-5663/& 2014 Elsevier B.V. All rights reserved.

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3.2. HRP-mimicking DNAzyme aptasensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemiluminescence aptasensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other optical aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Surface plasmon resonance aptasensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Surface-enhanced Raman scattering aptasensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Resonance scattering spectral aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Dynamic light scattering aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Ellipsometric aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Summary and conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. 5.

1. Introduction Biosensors are devices that transform the recognition of targets into a physically detectable signal, such as optical, electronic mass or magnetic signal. Generally, a biosensor contains two essentially functional components: target recognition and signal transduction. Theoretically, recognition component is the key for the sensor performance. An ideal recognition component should possess these characteristics of high sensitivity, admirable selectivity, fast response, robust performance and versatility for various targets. With these criteria, antibody and aptamer are two of the mostly used recognition component. Antibodies, responding to specific antigens, have been widely applied in the construction of various sensors. Enzyme immunoassays (EIAs) and enzyme-linked immunosorbent assays (ELISAs) are two of the most common methods. However, as a functional protein, antibody is sensitive to circumstance and its activity is unstable. What is more, it is difficult to obtain antibodies for molecules too small and molecules with high toxicity or poor immunogenicity, which limits its widespread application (Liu et al., 2009). Alternative, aptamer is single-stranded oligonucleotide with impressive recognition feature. As a strong rival to antibody, aptamer has high affinity and selectivity. The high affinity is attributed to the remarkable dissociation constants (Kd) ranging from picomolar to nanomolar levels between aptamer and its target (Brody et al., 1999). The excellent selectivity is stemmed from the reason that aptamer can distinguish even minor structural differences between targets and their analogs (Jenison et al., 1994). In addition, aptamers possess many other competitive advantages over antibody. Firstly, aptamers can bind with a broader range of targets, including metal ions, amino acids, other small organic molecules, viral proteins, even cells and bacteria (Ueyama et al., 2002; Harada and Frankel, 1995; Stojanovic et al., 2000; Koch et al., 2004; Bruno and Kiel, 1999; Wang et al., 2003). Secondly, aptamer can be successfully obtained from synthetic chemicals with the characteristics: higher purity and lower costs. Thirdly, aptamers can be flexibly modified with various chemical tags including fluorescence probes, electrochemical indicators and nanoparticles. They can also be modified with enzymes to minimize their degradation. Finally, aptamers are small in molecular weight and superior in stability, which can bear repetitious denaturation and renaturation. Overall, these unique characteristics make aptamers an ideal recognition element for biosensors. As to the signal transduction, electrochemical, optical, masssensitive transduction modes have been applied to biosensors (Cheng et al., 2009; Zuo et al., 2009; Zhou et al., 2010; Wen et al., 2011; Ruan et al., 2012; Shu et al., 2013; Liu et al., 2013; Huang et al., 2013; Kobu et al., 2013; Lim et al., 2010; Lu et al., 2011; Zeng et al., 2012; Fu et al., 2013; Iliuk et al., 2011). Among them, optical analysis has been widely developed because of high sensitivity,

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71 71 72 72 72 73 73 73 73 73 73 73

quick response and simple operation. Coupling aptamer as the recognition component with various optical analytical techniques as the signal transductions, optical aptasensor provides special opportunities for the analysis of targets. Up to now, numerous optical aptasensors have been developed for the detection of small molecules, proteins and metal ions (Zeng et al., 2012; Zhang et al., 2013; Liu et al., 2009). Specially, the quantitative detection of small biomolecules increasingly attracts much more interest of researchers due to its important physiological function. The normal amount of small biomolecule is crucial for organisms, while the amount too high or too low will cause certain diseases. For example, adenosine, an endogenous nucleoside, has potent antiarrhythmic effect and vasodilator activity. Its content also affects the peripheral and central nervous system (Wu et al., 2007). ATP, as energy currency in cells, is essential in living organisms. Its concentration is an important indicator for disease diagnosis such as cell injury and cell viability (Zeng et al., 2012). Therefore, the quantitative detection of small biomolecules is important in biomedical, diagnosis and treatment of diseases (Kerman et al., 2006; Shankaran et al., 2007; Chughtai and Heeren, 2010). To date, many optical aptasensors for various small biomolecules have been developed, such as ATP, adenosine, cocaine, dopamine, NAD þ , ochratoxin A, theophylline, flavin mononucleotide, tyrosinamide, kanamycin, oxytetracycline, glucose, bisphenol A (Li et al., 2012, 2013; Ye et al., 2013; Song et al., 2012; Fu et al., 2013; Zhou et al., 2011a, 2011b; Zheng et al., 2011; Lu et al., 2011; C. Yang et al., 2011; X.H. Yang et al., 2011; Galarreta et al., 2013; F. Li et al., 2009; M.J. Li et al., 2009; Chávez et al., 2010; Stojanovic and Kolpashchikov, 2004; Guieu et al., 2011; Song et al., 2011; Kim et al., 2010; Wang et al., 2013; Lee et al., 2011). All these analytes are listed in Table 1. Nevertheless, there are still few reviews focused on this field (Famulok, 1999; Walter et al., 2012). Moreover, they are mainly focused on the recognition of aptamer with its targets. A review on the development of these optical aptasensors for small biomolecules will give readers an overall understanding of its progress and provide some theoretical guidelines for its future development. Hence, we give a mini-review on the advance of optical aptasensors for small biomolecules, with emphasis not only on the recognition between aptamers and its targets but also on the design of signal transduction. In the following context, we will summarize these recent advances in optical aptasensors for the quantitative detection of small biomolecules. The context can be broadly divided into four categories: fluorescence aptasensors, colorimetric aptasensors, chemiluminescence aptasensors and other optical aptasensors.

2. Fluorescence aptasensors Fluorescence is one of the most common optical techniques and has been widely applied to aptasensors because of its unique

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Table 1 A tabulated summary of aptasors for small biomolecules. Analytical techniques

Classify

Subclassify

Targets

Detection limit

Ref.

Fluorescence

Covalent

FRET-based aptasensor

L-argininamide

– nM pM nM

Ozaki et al. (2006) Zeng et al. (2012) Lu et al. (2011) J.Q. Zhang et al. (2010), L.B. Zhang et al. (2010), X.B. Zhang et al. (2010) Sheng et al. (2011) He et al. (2011) Huang et al. (2010) Huang et al. (2011) He et al. (2010) Wang and Zhou (2008), Wang and Liu (2008) He et al. (2010) F. Li et al. (2009), M.J. Li (2009) Song et al. (2012) Cai et al. (2011) Chen et al. (2010) Zhou et al. (2011a, 2011b) Stojanovic and Kolpashchikov (2004)

Nanoparticle-based fluorescence aptasensor

Other labeled fluorescence aptasensors Label-free

Intercalators-based fluorescence aptasensors AP site based fluorescence aptasensors Metal nanomaterials-based fluorescence aptasensors Other label-free fluorescence aptasensor

Colorimetry

Au nanoparticles aptasensor

DNA-functioned AuNPs aptasensor Label-free AuNPs aptasensor

HRP-mimicking DNAzyme Chemiluminescence Surface plasmon resonance Surface-enhanced Raman scattering Resonance scattering spectral Dynamic light scattering Ellipsometry

characteristics: high sensitivity, high efficiency and simple operation. Fluorescence aptasensors can be mainly divided into two modes: labeled and label-free fluorescence aptasensors. 2.1. Labeled fluorescence aptasensors In nature, few biomolecules and aptamers are of autofluorescence. Label is necessary for a measurable signal. With the advantages of simple design and rapid detection process, the labeled fluorescence aptasensors for small biomolecules are the earliest reported technique (Wang et al., 1996). Fluorescence resonance energy transfer (FRET)-based aptasensors is a typical labeled fluorescence aptasensor. Recently, in view of the excellent quenching ability or fluorescence property of nanoparticles, the development of nanoparticles provides an alternative platform for labeled fluorescence aptasensors. In addition, taking use of some molecule's special fluorescence properties, the novel fluorescence aptasensors have also been developed. 2.1.1. Typical FRET-based fluorescence aptasensors As we all know, the aptamer will transform its conformation after binding with its target. Based on this principle, various aptasensors have been developed for small biomolecuoles (Ozaki et al., 2006; Li et al., 2008; Tang et al., 2008; Huang and Liu, 2010;

ATP NAD þ ATP

μM μM μM nM nM μM nM – μM μM μM μM –

Ochratoxin A ATP ATP Adenosine Cocaine ATP Cocaine Theophylline Adenosine ATP Adenosine Cocaine Flavin mononucleotide Adenosine Theophylline Tyrosinamide Kanamycin Dopamine Ochratoxin A Oxytetracycline AMP Cocaine Cocaine ATP Adenosine Cocaine Cocaine ATP Adenosine

μM – μM nM μM nM nM μM nM nM nM pM μM μM nM nM

ATP Adenosine Glucose Ochratoxin A Bisphenol A

nM – nM – fM

Fu et al. (2013) Chávez et al. (2010) Guieu et al. (2011) Song et al. (2011) Zheng et al. (2011) C. Yang et al. (2011), X.H. Yang et al. (2011) Kim et al. (2010) Teller et al. (2009) Du et al. (2011) Li et al. (2013) Li et al. (2013) Wang et al. (2011) Golub et al. (2009) S.J. Chen et al. (2008) Ye et al. (2013) J.Q. Zhang et al. (2010), L.B. Zhang et al. (2010), X.B. Zhang et al. (2010) Liang et al. (2011) C. Yang et al. (2011), X.H. Yang et al. (2011) Wang et al. (2013) Galarreta et al. (2013) Lee et al. (2011)

Y.X. Wang et al., 2010; Zeng et al., 2012). It has been demonstrated that the two split subunits of aptamer are still able to bind together with the aid of target (Huizenga and Szostak, 1995). Inspired by this phenomenon, Huizenga et al. developed an aptasensor for rATP and cocaine (Stojanovic et al., 2000). Later, single-chain aptamer has also been adopted in the design of conformational change aptasensors (Stojanovic et al., 2001; Ozaki et al., 2006). In their designs, the single-strand aptamer was turned into a hairpin after binding with its target, resulting in the proximity of fluorophore and quencher. Watson–Crick hydrogen bonds involved in these above aptasensors have some drawbacks, such as instability in complex environment and inflexibility (Y.X. Wang et al., 2010). To address this issue, Y.X. Wang et al. (2010) took the metal-ligation as the substitute of Watson–Crick hydrogen bonds to construct an aptasensor for ATP. In his design, two short C-rich DNA strand were labeled with a fluorophore and a quencher respectively. The two C-rich DNA were linked at the end of the aptamer to form the aptasensor. Without the target, the aptasensor formed a MB owing to the metal-ligation of C–Ag þ –C, leading to the quenching of fluorescence. Upon the adding of the target, the interaction between aptamer and its target opened MB structure and the fluorescence was recovered. Unfortunately, the sensitivity is unsatisfactory, which may derive from the fact that the metal-ligation C–Ag þ –C is not as stable as Watson– Crick bonds.

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Although the direct label to aptamers has made great progress in aptasensors for small molecules, the label of aptamer will more or less affect the binding affinity and selectivity of aptamer. As an alternative, some aptasensors based on molecular beacon (MB) have been developed (Li et al., 2008, 2007; Lu et al., 2011; Zeng et al., 2012). MB is a single-strand DNA with a fluorophore and a quencher at its ends. Ma et al. (2008) developed an aptasensor based on MB as the auxiliary signal probe. In his design, there were two oligonucleotide strands, oligo A and oligo B. With the aid of T4 polynucleotide kinase (PNK), oligo A with a 50 -hydroxyl group was phosphorylated by ATP. After phosphorylation, the oligo A and oligo B were linked together to open the MB assisted by Escherichia coli DNA ligase, giving rise to the recovery of fluorescence. The separation of target recognition and signal transport eliminates the label of aptamer and makes aptasensors more robust. Inspired by the above-mentioned design, various fluorescence aptasensors based on MB have been reported (Li et al., 2007; Lu et al., 2011; Zeng et al., 2012).

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Nevertheless, these above aptasensors exhibit moderate sensitivity. To improve the sensitivity, great efforts have been devoted to developing amplification approaches. For example, Shlyahovsky et al. (2007) took use of nicking enzyme-mediated strand displacement polymerase reaction to develop an amplification sensor for cocaine. X.B. Zhang et al. (2010) developed an amplified method based on the catalytic and molecular beacon (CAMB) for adenosine with high sensitivity. In their design, the binding of targets to its aptamer activated DNAzyme. Then, the activated DNAzyme cleaved and opened multiple MB strands to form the CAMB system. To further improve the sensitivity, Lu et al. (2011) constructed a double signal amplification platform based on the combination of split DNAzyme ligation reaction and CAMB. First, targets as the cofactor activated the DNA ligase and formed an integrated 8-17 DNAzyme. Subsequently, through the introduction of invasive DNA, the 8-17 DNAzyme was liberated from dsDNA and activated the CAMB system (Fig. 1). By means of signal amplification, the detection limit of these aptasensors for ATP or adenosine is

Fig. 1. Schematic illustration of a double signal amplification aptasensors based on split DNAzyme ligation reaction and CAMB for ATP and NAD þ . First, targets as the cofactor activated the DNA ligase and formed an integrated 8-17 DNAzyme. Then through the introduction of invasive DNA, the 8-17 DNAzyme was liberated from dsDNA and activated the CAMB system (Lu et al., 2011).

Fig. 2. Schematic illustration of an amplified graphene-based aptasensor for ATP. Without the targets, the labeled aptamer was absorbed onto the graphene with low fluorescence intensity. The binding of target and aptamer released aptamer from graphene. The aptamer/target complex was further digested by the nuclease and the liberated target could bind with another aptamer, thus leading to signal amplification (Lu et al., 2010).

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in pM level, which is two orders lower than that without amplification (Li and Ho, 2008; Zeng et al., 2012).

Lu et al. (2010) ingeniously developed a sensitive aptasensor for ATP based on the DNA protection of carbon nanomaterials and recycling of nuclease cleavage (Fig. 2). In his work, the aptamer was labeled with a fluorophore. Without the targets, the aptamer was absorbed onto the graphene and protected from digestion. Owing to the excellent quenching of the graphene, the background was low. In the presence of targets, the aptamer turned into its tertiary structure and was released from graphene. Then aptamer/ target complex was further digested by the nuclease and the liberated target could bind with another aptamer on the graphene, thus achieving signal amplification. Combining the low background with the signal amplification, a sensitive detection for ATP was achieved with a detection limit of 40 nmol/L.

2.1.2. Nanoparticle-based fluorescence aptasensors Stemming from their nanoscale dimension, carbon nanomaterials possess some remarkable properties, such as excellent quenching ability, selectively interaction with short single-stranded DNA (ssDNA), the ability of protecting DNA from the digestion of nuclease etc. These properties make them an ideal candidate for signal transduction in the development of sensing systems. Based on the excellent quenching ability of carbon nanomaterials towards fluorophore, gratifying results have been achieved (L.B. Zhang et al., 2010; He et al., 2011; Sheng et al., 2011). For example, L.B. Zhang et al. (2010) has developed a platform for ATP by using a coiled aptamer.The aptamer was labeled with a fluorophore. Without the target, the aptamer was in a coiled form and adsorbed onto the carbon nanotubes, leading to fluorescence quenching. Upon the addition of the target, the aptamer turned to its tertiary structure and was released from the carbon nanotubes, resulting in the recovery of its fluorescence. However, this design is only suitable for these targets whose bindings can induce a distinctive allosteric change of their aptamers, which limits its applications. To address this issue, Zhen et al. (2010) worked a split-aptamer aptasensor for ATP. Firstly, the aptamer was split into two subunits. One subunit was designed to form a MB and labeled a fluorophore at one end, while the other was in a coiled form. Without targets, the two subunits were absorbed onto the carbon nanotubes with low signal intensity. Upon the addition of ATP, the two subunits combined together to form a double-stranded DNA (dsDNA) and were released from carbon nanotubes, with the recovery of fluorescence.

2.1.3. Other labeled fluorescence aptasensors For a sensitive detection, high signal intensity and low background are two requirements. To data, different label methods have been reported with low background to improve overall sensitivity. Pyrene is a kind of spatially sensitive fluorescence dye. The signal intensity of pyrene monomers is negligibly, while two pyrenes in close will form a pyrene excimer with high fluorescence intensity. Besides, the excimer offers a large stokes shift (  130 nm) and long fluorescence lifetime ( 40 ns), while fluorescence lifetime of chromophores in biological fluids is usually less than 10 ns. Therefore, with time-resolved fluorescence measurements, the problem of interference background in biological media can be overcome. Wu et al. (2010) took use of pyrene and constructed an aptasensor for cocaine (Fig. 3). The aptamer was cleaved into two subunits and each one was modified with a pyrene moiety. The target brought the two subunits together and thus took the two pyrene moieties in close proximity, generating a

Fig. 3. Schematic illustration of an aptasensor for cocaine based on fluorescent molecular-pyrene (Wu et al., 2010). The aptamer was cleaved into two subunits with a pyrene moiety at the end. The target brought the two subunits together and took the two pyrene moieties in close proximity, resulting in the formation of a fluorescence exciter.

Fig. 5. A label-free aptasensor for cocaine based on strand displacement. The aptamer was split into two sections. Cocaine brought the two sections together and the primer sequence was exposed to trigger the strand displacement amplification. A label-free fluorescence signal was produced with the intercalation into SYBR Green I (He et al., 2010).

Fig. 4. Schematic illustration of an aptasensor based on home-made europium. The reporter DNA was labeled with europium. Without targets, the immobilized aptamer hybridized with reporter DNA and generated a high fluorescence signal. With the target, the aptamer preferred to binding with targets and released the reporter DNA, with the decrease in fluorescence intensity (Huang et al., 2011).

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fluorescence exciter. To avoid the label to aptamer, Huang et al. (2010) designed a novel aptasensor by the introduction of a reporter DNA. The reporter DNA was modified with pyrene molecules at both ends. Without targets, aptamer hybridized with the reporter DNA, making the two pyrene moieties far from each other. Upon the binding of targets with aptamer, the reporter DNA turned into a hairpin conformation, generating a fluorescence exciter. Besides pyrene, Huang et al. (2011) constructed a lowbackground aptasensor with home-made europium as signal output (Fig. 4). The aptamer was immobilized on the surface of glass slide. Then the reporter DNA modified with europium hybridized with the aptamer. Without targets, a high fluorescence signal was detected. Upon adding targets, the aptamer turned to bind with targets and released the reporter DNA. Profited from the large stoke shift and the long fluorescence lifetime of europium, this aptasensor exhibited low fluorescence background. 2.2. Label-free fluorescence aptasensors The label is usually time-consuming, labor-intensive and costeffective. Moreover, the label to aptamer will affect the binding affinity and selectivity of aptamer. Recently, label-free aptasensors have attracted growing interest, involving DNA intercalators, aptamer-binding dyes, abasic-site-binding dyes and metal nanomaterials. 2.2.1. DNA intercalators-based fluorescence aptasensors DNA intercalation dyes are one of the most common signal reporters for label-free fluorescence aptasensors. In aqueous solution, the dye molecule shows negligible fluorescence intensity, while strong fluorescence signal can be observed when they intercalate into dsDNA. Using the intercalation dye of (Ru (phen)2(dppz))2 þ , Wang et al. (2005) have achieved the quantitative detection for ATP in homogeneous solution. Later, Wang and Liu (2008) used a light harvesting cationic tetrahedralfluorene to further enhance the fluorescence intensity of ethidium bromide (EB). Benefited from tetrahedralfluorene, the detection limit for ATP is nearly 10-fold lower than that without tetrahedralfluorene. To further enhance the signal, He et al. (2010) introduced signal amplification strategy into the aptasensors for cocaine (Fig. 5). The cocaine aptamer was split into two sections, named as the hairpinprobe and single-stranded-probe (ss-probe). The presence of cocaine brought the two sections together and formed a tripartite complex. Then the exposed sequence in hairpin probe triggered strand displacement amplification. After amplification, a large amount of dsDNAs were produced and an obviously enhanced fluorescence signal was observed with the intercalation of SYBR Green I. The detection limit of cocaine was as low as 2 nmol/L. 2.2.2. Abasic-site-binding dyes-based fluorescence aptasensors An abasic or apurinic/apyrimidinic (AP) site is the depurine or depyrimidine site. Some fluorophores can selectively bind with the AP sites in dsDNA, accompanying with an obvious change in fluorescence intensity. This characteristic has makes AP site a potential candidate for nucleobase recognition and the detection of small molecules (Sankaran et al., 2009; Thiagarajan et al., 2009; Ong et al., 2009; Anderson and Mecozzi, 2005; M.J. Li et al., 2009). Xu et al. (2009a) and Xiang et al. (2009) designed a label-free aptasensor for adenosine based on AP site with a similar principle. In their design, the addition of targets released the AP sitecontaining antisense DNA from the dsDNA of aptamer/AP sitecontaining antisense DNA and thus liberated fluorophore to emit fluorescence. For a simple design, an aptasensor based on the selfassembly of aptamer was reported (Xu et al., 2009b). With assistance of targets, two fragments of aptamer were reassembled

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together. Then the fluorescence molecules bound into the AP site in the dsDNA. This design not only avoided the introduction of antisense DNA but also realized both “signal-off” and “signal-on” assays by using 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) and 3,5-diamino-6-chloro-2-pyrazine carbonitrile (DCPC) as signal reporters, respectively. To improve the sensitivity, Song et al. (2012) combined the label-free fluorescence aptasensors with CAMB signal amplification for a sensitive detection of adenosine (Fig. 6). Different with other CAMB system (X.B. Zhang et al., 2010; Lu et al., 2011), there was an AP site in the stem of MB for the label-free detection. At first, the fluorophore, ATMND, strongly bound with the AP site with low fluorescence intensity. Upon the binding of targets, the DNAzyme was activated and catalyzed the cleavage of MB, thus releasing ATMND with the recovery of its fluorescence. These above designs all require the modification of DNA with dSpacer or spacer C3, which is cost-effective and difficult for detection in the biological system. To address this issue, Xiang et al. (2010) put forward a general approach with unmodified DNA by replacing AP site with a vacant site. In his work, modificationfree fluorescence aptasensors for Pb2 þ , UO22 þ , Hg2 þ and adenosine were successfully developed. 2.2.3. Metal nanomaterials-based fluorescence aptasensors Recently, there have been ever-growing interests in the development of metal nanomaterials attributed to the following characteristics: facile synthesis, tunable emission, ideal photostability and small size. Cai et al. (2011) developed a silica nanoparticlesbased aptasensor for ATP. Firstly, the aptamer bound with its complementary DNA which was immobilized on silica nanoparticles. Then the signal reporter, Hoechst 33258, intercalated to the dsDNA for signal output. In the present of the ATP, the aptamer bound with ATP. Hoechst 33258 was released into solution and its fluorescence was quenched. Taking advantage of the property that

Fig. 6. A label-free and sensitive aptasensor for adenosine based on AP site and CAMB. At first, the fluorophore, ATMND, strongly bound with the AP site in MB. After the adding of adenosine, the DNAzyme was activated and started to cleave MB, releasing ATMND from the AP site, thus the fluorescence of ATMND was recovered (Song et al., 2012).

Fig. 7. A label-free and sensitive aptasensor for cocaine based on AgNCs. The aptamer was split into two sections and conjugated with a G-rich fragment. Cocaine brought the two sections together, thus putting the two G-rich sequence fragments in proximity. The G-rich sequence in proximity in turn greatly enhanced the fluorescence of AgNCs (Zhou et al., 2011a).

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Fig. 8. An amplified platform for adenosine based on DNA hairpin self-assembly. The binding of adenosine with aptamer released the inhibit strand and triggered the selfassembly of H1 and H2, resulting in the formation of G-quadruplex. With the interaction of NMM into G-quadruplex, high fluorescence signal was detected (Fu et al., 2013).

silica nanoparticles can protect DNA from digesting by enzyme, this aptasensor can achieve analysis in biological samples. Utilizing the excellence quenching nature of gold nanoparticles, Chen et al. (2010) developed an aptasensor for adenosine. The aptasensor contains the adenosine aptamer (AptAdo), gold nanoparticles (AuNPs) and the DNA-binding dye Oligreen (OG). The DNAbinding dye OG would bind with AptAdo in the form of AptAdo– OG with high fluorescence. Without adenosine, AptAdo–OG was absorbed onto the surface of AuNPs, resulting in the quenching of fluorescence. Upon the present of adenosine, the aptamer took a conformational switch from a coil to a G-quadruplex structure. The steric hindrance leaded to less absorption of AptAdo–OG onto AuNPs. Therefore, a high fluorescence was detected. The detection limit of adenosine is 70 nmol/L. To further improve the sensitivity, they creatively adopted AptPDGF, which had a higher affinity towards OG. Benefited from this property, the fluorescence intensity of the system with AptPDGF was twice over than that without AptPDGF. With AptPDGF for amplified fluorescence signal, the detection of adenosine was down to 5.5 nmol/L. It is reported that the dark Ag nanoclusters (AgNCs) could be converted into bright emitter when in proximity to guanine-rich DNA (Yeh et al., 2010). Based on this theory, Zhou et al. (2011a) constructed a one-pot, label-free aptasensor for cocaine (Fig. 7). Taking dsDNA as the formation template of Cu nanoparticles (CuNPs), this group built another aptasensor for cocaine detection (Zhou et al., 2011b).

2.2.4. Other label-free fluorescence aptasensors Malachite green (MG) is triphenylmethane dye with extremely low fluorescence in solution but strong fluorescence intensity (  200 fold) after binding with its RNA aptamer (Babendure et al., 2003). Taking advantage of its unique fluorescence property, Stojanovic and Kolpashchikov (2004) firstly designed a modular aptasensor for the determination of various analytes. The modular aptasensors consist of MG aptamer as signal domain, other aptamers (ATP, flavin mononucleotide or theophylline) as recognition domain and a communication module as a conduit between recognition and signal domain. The interaction of the target with its aptamer led to the exposure of the MG aptamer. Then enhances fluorescence intensity was detected after the binding of MG with its aptamer. Similarly, Xu and Lu (2009) reported an aptasensor for adenosine. The aptasensor consists of free MG, an aptamer strand

and a bridge strand. The aptamer strand is a chemical conjugate of the adenosine aptamer sequence and MG aptamer sequence. The function of bridge stand is to hybridize with aptamer stand. In the absence of adenosine, the bridge strand prevented MG aptamer from binding with MG. When adenosine was added into the system, the adenosine aptamers bound to its targets with the release of bridge strand. As a result, MG bound with its aptamer and emitted high fluorescence. These above aptasensors based on MG and its aptamer achieved a sensitive and selective detection for various targets. However, the aptamer of MG is RNA, which is unstable and expensive. Thus, the application of aptasensors based on MG is limited. Recently, our group developed a novel label-free and enzymefree fluorescence aptasensor based on target-catalyzed hairpin self-assembly for adenosine (Fig. 8) (Fu et al., 2013). This aptasensor contained aptamer-catalysis strand, inhibit strand, hairpin structures H1 and H2. Meanwhile, the G-quadruplex sequence was partly hidden in the stem of H2. Without targets, aptamercatalysis strand was inactivated because of the hybridization with inhibit strand. In the present of adenosine, the binding of aptamer with adenosine released the inhibit strand and activated catalysis strand. Then the catalysis strand triggered the self-assembly between H1 and H2, resulting in the formation of G-quadruplex. At last, N-methyl mesoporphyrin IX (NMM) was added to generate fluorescence signal.

3. Colorimetric aptasensors Colorimetry, as the simplest sensing mode, has been widely developed for aptasensors. Colorimetric aptasensors mainly contain Au nanoparticle aptasensors and HRP-mimicking DNAzyme aptasensors. 3.1. Au nanoparticles aptasensors Gold nanoparticles (AuNPs) have shown tremendous potential in colorimetric aptasensors, based on the phenomenon that the appearance color of dispersion state is different from that in aggregation state. AuNPs aptasensors can be divided into two categories: DNAfunctioned AuNPs aptasensor and label-free AuNPs aptasensor.

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3.1.1. DNA-functioned AuNPs aptasensors Liu and Lu (2004) firstly adopted DNA-functioned AuNPs for the colorimetric detection of small molecules. In their design, they combined the aptamer with DNAzyme together to construct the aptasensor. Without targets, the aptasensor was inactive and the substrate DNA could link the two short ssDNA attached on the AuNPs together, resulting in the aggregation of AuNPs. In the presence of targets, the interaction between aptasensor and target activated its cleavage activity towards the substrate DNA, thus dispersing the AuNPs. Later, to simplify the design, Zhao et al. (2007) and Chávez et al. (2010) achieved a colorimetric assay for adenosine and theophylline based on structure-switching aptamer and salt-induced aggregation. For a simple design, S.J. Chen et al. (2008) adopted the aptamerfunctioned gold nanoparticles for the direct detection of adenosine. In their design, the aptamer was attached on the AuNPs in a free coil and could not protect AuNPs from aggregation in the high salt solution. Upon binding with targets, the aptamer folded into a fourstranded tetraplex structure (G-quartet). Then AuNPs dispersed due to the steric hindrance and the increase in charge density of the AuNPs. This design with only one kind of DNA achieved a simple detection for adenosine. However, this design was only suitable for those targets whose binding can induce a special structure change of aptamer. Later, for a broad range of applications, F. Li et al. (2009) developed a cleaved-aptamer-based colorimetric aptasensor. They divided the aptamer into two subunits and attached them to different AuNPs. Without the targets, the AuNPs were dispersed. With the targets as the linker, the two subunits bound together, resulting in the aggregation of the AuNPs. 3.1.2. Label-free AuNPs aptasensors Nevertheless, these above methods need the function of AuNPs with thiolated DNA, which is a time-consuming process (1–2 days) (Wang et al., 2007). For a simple and rapid detection, many labelfree AuNPs-based aptasensors have been developed. The principle of label-free AuNPs-based aptasensors is show in Fig. 9. Although both ssDNA and AuNPs are negatively charged, the coordination interaction between the nitrogen atoms of the DNA and the AuNPs is stronger. Therefore, the unfolded aptamer can be adsorbed onto the AuNPs and prevents AuNPs from aggregating in salt. When aptamer turned into a target-aptamer complex, it will be released from the AuNPs. Then the addition of salt will lead to the aggregation of AuNPs. Based on this principle, many small molecules, such as tyrosinamide (Guieu et al., 2011), kanamycin (Song et al., 2011), dopamine (Zheng et al., 2011), Ochratoxin A (C. Yang et al., 2011), oxytetracycline (Kim et al., 2010), have been detected using unmodified AuNPs. However, these targets, which cannot induce an obviously change in its aptamer conformation, cannot be sensed by this aptasensor.

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As an alternative, Wang et al. (2007) developed a AuNPs aptasensor for ATP based on displacement strategy. The aptamer initially hybridized with ssDNA. The duplex in solution was too rigid to be absorbed onto AuNPs. Then, the adding of targets led to the dehybridization of the dsDNA. At last, the released ssDNA was absorbed onto the AuNPs and protected AuNPs from aggregation. Later, Zhang et al. (2008) developed a “divided aptamer” strategy to accelerate the detection process. The aptamer of cocaine was divided into two parts. Initially, the two parts was absorbed on the AuNPs and protected AuNPs from aggregation. Then cocaine took these two parts into a folded structure, resulting in the aggregation of AuNPs. 3.2. HRP-mimicking DNAzyme aptasensors HRP-mimicking DNAzyme is a kind of functional nucleic acid with the peroxidase-like activity with a G-quadruplex structure, which is generally stabilized by alkali metal cations (Sen and Gilbert, 1990). HRP-mimicking DNAzyme can effectively catalyze H2O2-mediated oxidation of 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt (ABTS) with an colorimetric output signal (Travascio et al., 1998). Li et al. (2007) designed a colorimetric aptasensor based on HRP-mimicking DNAzyme. The aptasensor consisted of the aptamer and the HRP-mimicking DNAzyme. A block DNA was added to inhibit the activity of DNAzyme. With target, the aptasensor turned its conformation to bind with the target and the block DNA was released due to the less stability of the dsDNA. At last, the liberated DNAzyme recovered its activity and gave a colorimetric signal. Later, Elbaz et al. (2008) developed a parallel analysis method for two targets by using a bifunctional aptasensor. The bifunctional aptasensor consisted of a long aptamer and HRP-mimicking DNAzyme. The long aptamer was constructed by cocaine aptamer and AMP aptamer. DNAzyme acted as a block chain and hybridized with the long aptamer. Then either of the two targets activated the aptasensor, generating an output signal. Thereafter, in order to simplify design, Teller et al. (2009) extended this paradigm of sensing and developed aptamer-DNAzyme hairpins for the amplified detection of AMP and lysozyme . These above aptasensors are all based on ABTS for a detectable signal. However, the oxidation product of ABTS is not stable and can quickly decay to colorless in aqueous media. To address this drawback, Du et al. (2011) reported a simple and sensitive colorimetric aptasensor for cocaine by using 3,3,5,5-tetramethylbenzidine sulfate (TMB) as the substrate of DNAzyme (Fig. 10). What is more, they combined the low background stemmed from magnetic separation with the high signal benefited from DNAzyme for a high sensitivity.

4. Chemiluminescence aptasensors

Fig. 9. Schematic representation of label-free AuNPs-based aptasensors. The unfolded aptamer was adsorbed onto the AuNPs and prevented AuNPs from aggregating in high salt solution. After reacting with its targets, aptamer released from the AuNPs, leading to the aggregation of AuNPs.

Chemiluminescence (CL) has shown tremendous potentiality and versatility in analytical application as a robust detection method. Different from other optical methods, the excitation energy of CL is from chemical reaction. The unique characteristics of CL contain high sensitivity, wide calibration ranges and simple instrumentation. Several CL aptasensors for small biomolecules have been successfully developed (Yan et al., 2009; Yan et al., 2010; Qi and Li, 2011). For example, Yan et al. (2009) achieved a label-free CL detection for adenosine. In their work, they adopted a special CL reagent, 3,4,5-trimethoxyl-phenylglyoxal (TMPG) as the signaling molecule, which reacted specially with guanine (G) nucleobases. Firstly, the capture DNA was attached on the surface of magnetic beads and hybridized with the G-rich adenosine aptamer.

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Fig. 10. The illustration of colorimetric sensor based on G-quadruplex DNAzyme. The aptamer was split into two sections. One of them was attached on the magnetic nanopaticles. The other one was conjugated with a G-quadruplex sequence. Cocaine brought the two sections together. After magnetic separation, high colorimetric signal was detected (Du et al., 2011).

After magnetic separation, the interaction between TMPG with G-rich aptamer generated a high chemiluminescence signal. Then the adding of target induced the release of G-rich aptamer and low chemiluminescence intensity was detected. This method is only applicable to G-rich aptamers, which limits its general application. As an alternate, new aptasensors based on chemiluminescence resonance energy transfer (CRET) were reported (Zhang et al., 2009; Jin et al., 2011; Zhou et al., 2012). In Zhang et al.'s design, they constructed a binary molecular beacon probe (BMBP) and a triplex DNA molecular beacon probe (TMBP) as signaling probes. A low background was detected. In contrast to FRET, CRET occurs by the oxidation of a luminescent substrate without an excitation source and eliminates the consideration of autofluorescence from biosystems and photobleaching of fluorescence dyes. The detection limit of BMBP and TMBP for ATP is 1.1  10  7 mol/L and 3.2  10  7 mol/L respectively. HRP DNAzyme, with the activity that catalyze some chemical reagents for amplified optical signal, has been applied to the CL aptasensors (Freeman et al., 2011; Li et al., 2011, 2013; Liu et al., 2011). Freeman et al. (2011) developed a CL aptasensors based on the HRP DNAzyme and split aptamer subunits. At first, the aptamer and the DNAzyme were split into two subunits and each aptamer unite was linked with a DNAzyme unite. One of the aptamer was attached on the quantum dots (QDs). The addition of ATP brought the units of aptamer together and thus formed an activated DNAzyme. With QD, a CRET signal was detected. Later, based on the phenomenon that some aptamer could be folded into the G-quadruplex with its targets, this group reported a new CRET-based aptasensors (Liu et al., 2011). Recently, Li et al. (2013) designed a sensitive CL aptasensor based on strand displacement amplification and HRP DNAzyme catalytic reaction for a further improvement in sensitivity.

5. Other optical aptasensors Besides fluorescence, colorimetry and chemiluminiscence, other optical analysis methods have also been used in aptasensors for small biomolecules, such as surface plasmon resonance detections, surface-enhanced Raman scattering detections, resonance scattering detections, dynamic light scattering detections and ellipsometry. In the following sections, we will focus on the progress of these optical aptasensors for small biomolecules. 5.1. Surface plasmon resonance aptasensors Surface plasmon resonance (SPR) is based on the change in refractive index which is proportional to the mass of biomolecules

on the SPR surface. Although SPR has been widely used in biological analytes (Piliarik and Homola, 2006; Piliarik and Homola, 2009), it is still a challenge for its detection of small biomolecules, which is because that the binding of small molecules with its aptamer causes too little change in refractive index for detection. As a solution, surface-enhanced plasmon resonance has been developed with the enhancement effect of metal nanostructures. Some works for small molecules have been reported based on the surface-enhanced plasmon resonance and an indirect competitive inhibition assay (ICIA) (Wang and Zhou, 2008; Wang et al., 2009). In their designs, the aptamer was first hybridized with its complementary DNA. Then targets competitively bound with aptamer with a higher affinity. At last, the dissociation of aptamer and its complementary DNA made functionalized metal nanoparticle far or near the SPR gold film, resulting in a changed SPR signal. Based on a similar principle, some SPR sensors based on split aptamer subunits have been developed (Golub et al., 2009; Wang et al., 2011). In their designs, the aptamer was divided into two subunits labeled with Au-NPs or attached to Au film. Upon the adding of targets, the two aptamer subunits bound together and resulted in the close proximity of Au-NPs and Au film or lead to nanoparticles aggregation, thus generating a distinct SPR signal change. Besides the methods based on split aptamer subunits, Wang et al. developed a SPR aptasensor by using the special conformational change of the adenosine aptamer (J. Wang et al., 2010). After binding with adenosine, the aptamer changed its conformation into a four-stranded tetraplex structure (G-quartet), which induced the side-by-side self-assembly of gold nanorods (AuNRs) with a change in SPR signal intensity. 5.2. Surface-enhanced Raman scattering aptasensors Surface-enhanced Raman scattering (SERS) relies on the principle that the Raman scattering intensity of molecules will be greatly improved after adsorbed onto the metal surface. Chen et al. (2008a) constructed a SERS aptasensor for cocaine by the split aptamer unites. Latter, to free aptamers from label, they developed another SERS aptasensor based on structure-switching signaling aptamer (Chen et al., 2008b). However, these methods possess some drawbacks: poor reproducibility, poor water-solubility and unfavorable for surface bioconjugation (Li et al., 2012). To overcome these shortcomings, Li et al. (2012) worked a new aptasensor for the detection of ATP using gold nanostar@Raman label@SiO2 core–shell nanoparticles as the SERS probe. Firstly, the aptamer with the SERS probe was hybridized with the ssDNA attached on the gold film, thus generating an enhanced SEPS signal. Upon the adding of ATP, the aptamer turned to bind with ATP and was

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released from the substrate with a reduced SEPS signal. Recently, for a sensitive detection, Ye et al. (2013) developed an amplified SERS aptasensor for small molecules based on primer self generation and strand-displacement polymerization. The combination of high signal output stemmed from signal amplification and low background derived from magnetic separation achieved an ultrasensitive detection for ATP with a detection limit of 0.05 nmol/L. 5.3. Resonance scattering spectral aptasensors Resonance scattering (RS) spectral method, with simplification and rapidness, has also found its application in small biomolecule aptasensor. J.Q. Zhang et al. (2010) developed an aptasensor for adenosine based on resonance scattering of gold nanoparticles. Without adenosine, the aptamer hybridized with the G-rich DNA attached to the AuNPs. The rigid dsDNA protected the AuNPs from aggregation. Then the adding of adenosine switched its aptamer from the dsDNA and the released G-rich DNA turned into a G-quadruplex, which induced the aggregation of AuNPs and gave an enhanced RLS signal. The detection limit of this method for adenosine is 1.8 nmol/L. To further improve the sensitivity, Liang et al. (2011) took use of the AuNPs-catalytic reaction of glucose– Cu(II) for the detection of ATP. Firstly, the aptamer was hybridized with a short ssDNA and the rigid and unbounded dsDNA could not protect the AuNPs from aggregation. With ATP, the aptamer preferred to binding with ATP. Then, the coil ssDNA was attached on the surface of AuNPs due to the electrostatic interaction and thus protected AuNPs from aggregation. Finally, the dispersive AuNPs catalyzed the reaction of glucose–Cu (II) with amplified RS signal output. The detection limit of ATP is 0.5 nmol/L. 5.4. Dynamic light scattering aptasensors Dynamic light scattering (DLS), based on the Brownian motion of molecules, is another optical analytical method for detection of small biomolecules. X.H. Yang et al. (2011) reported a dynamic light scattering aptasensor for adenosine based on the split aptamer subunits. In their work, the aptamer was split into two subunits and was attached to the surface of gold nanoparticles. The binding of adenosine with its aptamer brought the two split subunits together and took gold nanoparticles in proximity with a change in DLS signal. Later, Wang et al. (2013) designed a sensitive DLS aptasensors for glucose based on mercaptophenyl-boronic acid functionalized gold nanoparticles (MPBA-AuNPs). Without glucose, the MPBA-AuNPs were stable and water soluble in dispersed state. Due to the interaction of glucose with boronic acids on the MPBA-AuNPs, MPBA-AuNPs aggregated and generated changed DLS signal.

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6. Summary and conclusion In this review, we have comprehensively summarized the development of aptasensors for small biomolecules with various optical analysis strategies. Each optical analysis method possesses its unique characteristics. Fluorescence, colorimetry and chemiluminescence are three of the most common methods for aptasensors. Fluorescence is one of the most prevalent signal transduction modes, including labeled mode and label-free mode. In the labeled aptasensors, we introduce FRET-based aptasensors, nanoparticle-based aptasensor and other labeled aptasensors. In the label-free design, there are not only fluorescence molecules with special fluorescence properties (NMM, ATMND), but also novel nanomaterials (Ag NCs, CuNPs). Colorimetric detection represents the simplest sensing mode and can be visible to the naked eye. Thus, many colorimetric aptasensors for small biomolecules are widely designed, mainly involving AuNPs and the formation of HRP-DNAzyme with hemin. Chemiluminescence has also been exploited widely in recent years due to its wide calibration ranges and simple instrumentation. In addition, other optical analysis methods, including surface plasmon resonance, surface-enhanced Raman scattering, resonance scattering, dynamic light scattering and ellipsometry have also been recommended.

7. Future perspectives Various optical aptasensors have been successfully developed for small biomolecules. However, few of them can be applied in the biological samples. It is resulted from two aspects. On one hand, the concentration of some analysts in biological samples is too low for detection. On the other hand, the complex environments of biological samples will interfere with detection, even resulting in false positive signals. Therefore, great efforts have been devoted to improving the selectivity and sensitivity of optical aptasensors for small biomolecules. Fortunately, there are two of the most promising areas for the future optical aptasensors of small biomolecules. Firstly, the development of nanoscience and nanotechnology has promoted the appearance of nanomaterials with novel optical properties. These nanomaterials will provide new and unique insights for optical aptasensors. Secondly, the nature of aptamer is nucleotide. Therefore, many nucleic acid amplification technologies are potential to be applied in aptasensors for ultrasensitive detection, such as PCR, RCA, SDA, HCR and NEMA. Overall, the future of optical aptasensors possesses great potential with a brighter future, yet many hurdles remain to be overcome.

5.5. Ellipsometric aptasensors

Acknowledgments

Ellipsometry, based on the change in polarization, is a useful optical analytical technique. It can be used to investigate the properties of thin films, such as roughness, thickness, crystallinenature, doping concentration, electrical conductivity, etc. Some biosensors based on ellipsometry have been successfully developed (Balamurugan et al., 2006; Davis et al., 2009; Chlpík et al., 2013). For example, Siegel et al. (2009, 2014) carried out a series of works in regard to cardiovascular disease based on ellipsometric biosensors, including the determinations of small molecule biomarkers such as creatinine and prostaglandin. However, the application of ellipsometry in the aptasensors for small molecules is still a challenge. Galarreta et al. (2013) reported an aptasensor for ochratoxin A assisted by ellipsometry. In their work, the aptamer was immobilized on the surface of gold nanostructures. After the adding of ochratoxin A, the aptamer transformed its form from coil to specific tertiary structure. Then, they adopted the change in ellipsometry to evaluate the aptasensor.

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