Biosensors
Fluorescence Sensing
Molecular interactions are commonly detected using fluorescence technologies. AuNPs can serve as excellent fluorescence quenchers through fluorescence resonance energy transfer or an electron transfer mechanism,[41] and aptamers can be readily modified with fluorescence tags. Therefore, Apt–AuNPs should offer excellent fluorescence sensing. For example, our group developed an Apt–AuNP platform to measure the distance between two binding sites on a membrane receptor in live CEM cells.[42] Excess thiol-modified sgc8 and Alexa Fluor® 488 (Life Technologies, CA, USA) were modified on the AuNPs and the anti-PTK7 antibody through the primary amines on the heavy chain, respectively. The colocalization of sgc8 and anti-PTK7 to their individual binding sites on the PTK7 receptor brought the Alexa Fluor 488 and AuNPs into a fixed distance apart. When the donor dye molecule and acceptor AuNPs surface reach close proximity, quenching of fluorescence from the cell surface results. Therefore, by controlling the size of the AuNPs, the distance from the fluorophore molecule to the surface of AuNPs could be manipulated, given that the distance from the fluorophore on the antibody binding site to the center of the nanoparticle on the aptamer is equivalent to the distance between the two binding sites on the PTK7 receptor (Figure 3).
Figure 3.
Fluorescence sensing for measuring the distance between two receptor PTK7 binding sites on a live cell membrane.
AuNP: Gold nanoparticle; d: Diameter; R: Distance from the Alexa Fluor® 488 on the antibody-binding site to the center of the AuNP on the aptamer; r: Distance between the AuNP and Alexa Fluor 488 (when 'r' reaches a fixed number it results in the quenching of fluorescence).
Reproduced with permission from [42].
Mirkin et al. developed aptamer nanoflares to detect ATP inside living cells and, by reporter fluorescent signaling, quantified its concentration.[43] To accomplish this, AuNPs were functionalized with a dense monolayer of ATP aptamers hybridized with short complementary Cy5-labeled reporter strands. Once the nanoflares met ATP inside living cells, the aptamer interacted with its target, leading to a conformational change of the aptamers that displaced the Cy5-labeled strand to recover the fluorescence signal whose intensity could then be used to quantify intracellular ATP concentration.
Huang et al. developed a PDGF assay using Apt–AuNPs.[44] In this case, the fluorophore N,N-dimethyl-2,7-diazapyrenium dication can be intercalated with the PDGF aptamer, which has a unique triple-helix conformation such that the Apt–AuNP composites bring the AuNPs into close proximity with N,N-dimethyl-2,7-diazapyrenium dication, thereby quenching fluorescence. However, when binding with its target PDGF, the aptamer changes its conformation to prevent N,N-dimethyl-2,7-diazapyrenium dication intercalation and, in turn, induces the recovery of fluorescence.
Fan and coworkers reported novel multicolor fluorescent Apt–AuNPs for homogeneous detection of small-molecule targets.[45] First, the multicolor dye-labeled aptamers hybridize with DNA strands on the AuNPs, resulting in fluorescence quenching of the dyes. Then, in the presence of different target molecules, the dye-labeled aptamers interact with their target molecules, leading to fluorescence recovery of the dyes such that adenosine, potassium ion and cocaine can be simultaneously detected with high selectivity.
Colorimetric Sensing
AuNPs possess a strong SPR band that results from the spatial length reduction of electronic motion and the coherent oscillation of the electrons.[46] In addition, the SPR band of AuNPs has strong distance-dependent properties, whereby the aggregation of AuNPs of appropriate sizes (diameter >3.5 nm) can induce interparticle surface plasmon coupling, resulting in a visible color change from red to blue.[47] Thus, AuNPs provide a practical platform for colorimetric sensing of any target analyte. The key to a successful AuNP-based colorimetric sensing platform is control over the dispersion and aggregation of colloidal AuNPs. This could be accomplished by using biological processes of interest, which are guided by interparticle attractive and repulsive forces. Colloidal parameters, such as surface charges, including charge amount and density, and surface-grafted polymers, including molecular weight, graft density and conformations, are important factors for dispersion and aggregation.[48] Although not as sensitive as fluorescence sensing, colorimetric detection is a convenient alternative technique for analyte detection with the naked eye. Furthermore, the extinction coefficient of AuNPs is over 1000-times higher than that of organic dyes, thus enhancing the sensitivity of AuNP-based colorimetric detection. By combining aptamer-based analytical methods and an AuNP-based platform, colorimetric detection of small organic molecules has been used.[49–55]
In 1996, Mirkin et al. demonstrated a DNA-mediated AuNP assembly,[56] which later led to the extensive use of oligonucleotide-directed AuNP aggregation for colorimetric detection of oligonucleotides.[57–65] In this approach, two ssDNA-modified AuNP probes designed to be complementary to both ends of the target oligonucleotides were used for colorimetric detection. In the presence of a target, two AuNP probes hybridize with the two ends of the target, resulting in AuNP aggregation and color change.
Lu et al. demonstrated a general design for a colorimetric sensor system composed of three functional components: two kinds of ssDNA-modified AuNPs and a linker DNA molecule that carries an adenosine aptamer (Figure 4).[49] Initially, the two kinds of AuNP hybridized with linker DNA, resulting in aggregation of the AuNPs in solution to produce a purple color. In the presence of adenosine, the aptamer changed its structure to bind with it, resulting in the disassembly of the AuNP aggregates with a concomitant blue-to-red color change. Furthermore, by using a cocaine aptamer, Lu and coworkers have constructed a colorimetric sensor for cocaine to demonstrate the generality of this system.[49]
Figure 4.
Colorimetric sensing to measure adenosine.
The A12 in 3'-AdapAu denotes a 12-mer polyadenine chain. In a control experiment, a mutated linker with two mutations, as shown by the two red arrows, was used.
Reproduced with permission from [49].
Huang et al. constructed PDGF-binding Apt–AuNPs for sensitive detection of PDGF.[44] PDGF with detection limits of 3.2 nM could be detected by monitoring the changes in color and extinction of Apt–AuNPs that occurred as a result of aggregation.
Fan et al. developed a simplified colorimetric approach using unmodified AuNPs.[66] First, the target-free aptamer is hybridized with its complementary sequence, forming a rigid duplex. In the presence of the specific target, the aptamer binds to the target and changes its conformation by disassembling the original duplex and releasing a ssDNA strand, which, in turn, causes the unmodified AuNPs to aggregate, thus allowing colorimetric detection.
Wang and colleagues reported a dot blot assay for the detection of thrombin.[67] The protein was first immobilized on nitrocellulose. A color change from colorless to red was observed when the Apt–AuNPs bound to the active site of the protein on the membrane. The Apt–AuNPs could also be enhanced by silver, with a detection limit of 14 pM.
Our group developed a direct colorimetric assay for the detection of cancer cells by assembling AuNPs on cell membrane surfaces via recognition between aptamers and their target proteins, causing the SPR bands of the AuNPs to overlap and, in turn, providing direct visualization of different types of cancer cells.[68] Similarly, Zeng and colleagues reported a biosensor for the sensitive detection of circulating Ramos cells.[69] With this biosensor, 4000 Ramos cells were detectable with the naked eye, while as few as 800 Ramos cells could be detected by a portable strip reader.
Electrochemical Sensing
The excellent conductivity, high surface area and catalytic properties of AuNPs make them prime candidates for the electrochemical detection of a wide range of analytes. Owing to its high sensitivity, fast response and robustness, electrochemical detection based on aptasensors has attracted increasing attention. From as early as the 1990s, aptamers have been used as biorecognition elements for electrochemical biosensors.[70,71]
Kerman and colleagues reported a sandwich assay for thrombin analysis using Apt–AuNPs in which the primary and secondary aptamers were immobilized on the surface of a screen-printed carbon electrode and AuNPs, respectively.[72] The electrochemical reduction current response of AuNPs was monitored for the quantitative detection of thrombin with a limit of detection as low as 1 nM. To improve detection sensitivity, Li et al. fabricated an Apt–AuNP sensing system, in which they first immobilized the thiolated aptamers on a gold substrate to capture the thrombin molecules and then used the aptamer-functionalized AuNPs to amplify the impedimetric signals.[73]
Zhang and colleagues reported an electrochemical sensing strategy for highly sensitive detection of small molecules with a detection limit as low as 0.18 nM based on switching structures of aptamers.[74] First, a gold electrode was modified with AuNPs and a thiolated capture probe was immobilized onto the electrode via sulfur–gold affinity. Then, a linker DNA containing an antiadenosine aptamer sequence and reporter DNA were loaded onto the AuNPs. In the presence of adenosine, the aptamer bound adenosine and changed its structure. As a result, the reporter probes, together with the AuNPs, were released into solution and induced different decreases in peak current with the introduction of adenosine at different concentrations.
Based on three partially complementary ssDNA molecules, Kuang and colleagues developed an electrochemical aptamer-based sensor for ochratoxin A (OTA), which is known to be hepatotoxic, nephrotoxic, teratogenic and mutagenic to a wide variety of mammalian species (Figure 5).[75] First, one end of the OTA aptamer was hybridized with a linker oligonucleotide (DNA 1) which was immobilized on a glassy carbon electrode, while the other end of the aptamer was hybridized with an AuNP-functionalized oligonucleotide (DNA 2), which was used to amplify the sensing signal. In the presence of OTA, the aptamer bound with its target and released DNA 2, affecting the amount of DNA immobilized on the electrode surface. Methylene blue was used as the electrochemical probe and was proportional to the amount of DNA; consequently, the redox currents of the electrochemical probe methylene blue were proportional to the amount of OTA in the solution.
Figure 5.
Electrochemical sensing to measure ochratoxin A.
The MB is proportional to the amount of DNA 2, while the redox currents of the MB are proportional to the amount of OTA in the solution.
AuNP: Gold nanoparticle; MB: Methylene blue; OTA: Ochratoxin A.
Reproduced with permission from [75].
Furthermore, In order to enhance the sensitivity, Yuan et al.[76] and Kwak et al.[77] have successfully developed a system based on the sandwich assay by using the amplification of Apt–AuNP–horseradish peroxidase conjugates and enzymatic silver deposition to detect thrombin.
Electrochemical detection based on Apt–AuNPs offers many advantages, including high sensitivity and selectivity, compatibility with novel microfabrication technologies, low cost, disposability, simplicity of operation, and robustness. In the future, we are certain to see more growth in electrochemical detection.
Chemiluminescence Sensing
Chemiluminescence (CL) is known as a powerful and important analytical technique, and luminal–H2O2 is the most popular CL system. It needs no external light source, such as fluorescence, to produce a light signal, and it offers simplicity, low cost and extremely high sensitivity. Based on their large surface area and special structure, nanomaterials have recently been used to improve the sensitivity and stability of CL systems.
Cui's group found that gold colloids with nanoparticles of different sizes could enhance the CL of the luminal–H2O2 system based on the catalysis of AuNPs.[78] Qi and colleagues reported a label-free, aptamer-based CL system based on Apt–AuNPs and the catalytic activity of unmodified AuNPs on the luminal–H2O2 reaction.[79] Upon target binding, the aptamer induces the aggregation of AuNPs and further enhances the luminal–H2O2 CL reaction. Thrombin was detected as low as 26 fM, which is almost four orders of magnitude better sensitivity than any current AuNP-based colorimetric method. Another protein, R-fetoprotein, was detected with a 5 pg/ml limit of detection, which is much lower than the classic ELISA. Finally, Liu and colleagues reported an aptamer-based sandwich approach combined with the CL analysis to capture and detect rare cells on a microfluidic chip (Figure 6).[80] First, aptamers were immobilized on microfluidic channels to capture and isolate the specific cells from a cell mixture. Next, the AuNPs modified with aptamers were added to bind with the cells and trigger the CL reaction. The number of target cells was observed with a low detection limit of 30 target cells in a 3 µl cell mixture.
Figure 6.
Chemiluminescence sensing to measure rare cells.
The rare cells are captured by the aptamer and detected by chemiluminescence.
PDMS: Polydimethylsiloxane; PMT: Photomultiplier tube; SH: Sulfhydryl.
Reproduced with permission from [80].
Nanomedicine. 2013;8(6):983-993. © 2013 Future Medicine Ltd.
