Antibody-based nanotechnology

The rapid and precise identification of biomarkers (factors measured or observed that serve as indicators of normal biological or pathogenic processes) is crucial for early detection, monitoring, and indeed treatment of many life-threatening diseases. A wide range of biomarkers have been proposed in recent years for early disease diagnosis and monitoring of disease progression; these include circulating tumor cells (CTCs) [49], microRNAs [50], and certain enzymes [51]. For example, the quantification of protein biomarker levels in serum has been reported to hold promising potential for early cancer detection and treatment monitoring [52]. Nonetheless, despite considerable R&D, there is still an unmet need for the development and implementation of novel and accurate diagnostics tools. The integration of Abs as the detecting agents in nanobiosensor diagnostic devices offers several advantages, including high sensitivity and specificity. These properties are particularly important in the diagnosis of life-threatening diseases, where false positive or false negative results could compromise therapeutic strategies in the clinical setup. These considerations are currently propelling the development of Ab-based nanobiosensors.

Ab nanobiosensors are composed of two essential moieties, namely, a component that facilitates the detection of the analyte of interest with high specificity and a component for transducing the detection into a readable signal. In some instances, an amplification step is also required. A promising type of Ab-based nanobiosensor is based on measuring an electrochemical interaction between the Ab and the analyte: The induction of Ab-antigen complex conformational changes produced by the specific biorecognition between an Ab and an antigen generates a measurable signal. This approach provides high sensitivity and selectivity [53] and requires relatively simple instrumentation. In recent years, electrochemical nanobiosensors have been widely investigated for the detection of numerous analytes by integrating different electrochemical techniques and different nanomaterials. Such nanobiosensors are based on two types of Ab, namely, a detection Ab (tracer Ab) and a primary Ab (capture Ab). The capture Ab is designed to bind the target with high specificity and affinity, while the tracer Ab is labeled with a component that generates a measurable signal; such components are usually electrochemically active molecules, such as enzymes, metal nanoparticles, or quantum dots. The binding of the tracer Ab to the capture Ab enables the selective production of an electrochemical signal for the detection of a specific analyte.

Jin et al, for example, produced multi-nanomaterial electrochemical biosensors based on magnetic beads, graphene, and gold nanoparticles to detect an antigen (such as a cancer biomarker) with a rapid response time and high sensitivity, specificity and stability [54]. In this approach, the authors first coated magnetic beads with a capture Ab specific to the analyte to be detected. Specifically, they used an Ab that detected the model protein, carcinoembryonic antigen (CEA), which is elevated in the serum of cancer patients. Next, the coated magnetic beads were attracted to isolated monographene sheets by an external magnetic field, followed by the addition of gold nanoparticles functionalized with horseradish peroxidase (HRP) and a tracer Ab that recognized the CEA analyte. The anodic peaks generated by the catalysis of H₂O₂ by HRP represented the levels of the analyte in the measured sample. Using this sandwich-type immunoassay configuration, the authors demonstrated that the multi-nanomaterial electrode that was based on two different antibodies could detect the cancer-related biomarker CEA with rapid response and recovery times that were shorter than those of traditional strategies. Moreover, the detection limit was found to be 5 ng ml⁻¹, which meets the requirements for clinical diagnosis. However, the authors did not elaborate on the identity of the Abs that were used (monoclonal or polyclonal), which may affect the specificity of the system and the generation of either false positive or false negative results.

Zhang et al developed a simple, enzyme free, label-free controlled release system based electrochemical immunoassay (CRECIA) for the quantitative detection of brevetoxin B (PbTx-2) as a model compound [55]. PbTx-2, a neurotoxin produced by algae, disrupts normal neurological processes and causes the illness known as neurotoxic shellfish poisoning. The detection was based on target-responsive controlled release of methylene blue trapped in the pores of a polystyrene-microsphere-gated mesoporous silica nanocontainer to which monoclonal mouse anti-PbTx-2 Ab had been conjugated. The nanocontainer was opened upon the introduction of the target PbTx-2 analyte due to displacement of the negatively charged mAb-functionalized nanocontainer from the positively charged microspheres. This reaction resulted in the release from the pores of methylene blue, which was quantitatively monitored by square wave voltammetry. The demonstrated CRECIA approach offers a promising scheme for the development of an advanced homogeneous immunoassay without the need for sample separation and washing procedures. This approach also demonstrates how Abs can be utilized not only as capture/tracer molecules but also as inducers in molecular gate mechanisms.

Venkatraman et al investigated nanobiosensors based on the formation and perturbation of an electrical double layer (EDL) formed at a liquid/metal interface [56]. The analyte binding to the mAb-saturated nanowires at the interface induced a perturbation in the EDL and accordingly generated a change in the capacitance of the EDL. The assay was implemented for the detection of two biomarkers for cardiovascular disease, namely, the inflammatory proteins, C-reactive protein (CRP) and myeloperoxidase (MPO). In a similar way, Chang et al developed an assay for the detection with a flexible carbon-nanotube-based nanobiosensor of human serum albumin (HSA) [57], which is frequently used to monitor liver function [58]. The advantages conferred by the use of carbon nanotubes included a high surface-to-volume ratio, excellent electrical conductivity, and good mechanical strength, all of which are useful in the production of highly sensitive nanobiosensors. The carbon nanotubes were grown directly on a polyimide flexible substrate, and anti-HSA mouse IgG Ab was covalently bound to the carbon nanotubes. Bovine serum albumin (BSA) was applied to block non-specific binding and to enhance the detection specificity for HSA. HSA concentration was quantified using electrochemical impedance spectroscopy, with a very low detection limit.

Hu et al showed how a multi-component Ab-based nanobiosensor could serve as an accurate alternative to the conventionally used ELISA for the detection of urinary retinol-binding protein (RBP), a biomarker for renal tubular injury. The researchers developed a method in which anti-RBP mAb was immobilized on silver microspheres coated with BSA [59]. They found that coating the silver particles with BSA increased the surface area and provided good stability and, more importantly, outstanding biocompatibility. Using this approach, they demonstrated that the increased surface area of these microspheres facilitated an increase in the amount of bound Ab and, in turn, of the analyte immobilized on the modified electrode, while maintaining the bioactivity of the immobilized biomolecules. Moreover, the BSA-Ag coated particles showed good electric conductivity and improved the electrochemical sensing capability.

In recent years, the use of optical nanobiosensors has become increasingly prevalent by virtue of their outstanding detection performance; in particular, they offer label-free techniques with high sensitivity and real-time monitoring capabilities. This type of technology is based on monitoring changes in the refractive index in the proximity of the sensor surface. One such approach was applied to enhance a localized surface plasmon resonance (LSPR) bioassay signal by using plasmonically active gold nanoparticles conjugated to anti-biotin Ab. Upon binding of the anti-biotin Ab to its biotin target, an amplification of up to 400% of the wavelength shift was obtained [60], thereby improving the observed binding constant (by 2 orders of magnitude), the limit of detection (by nearly 3 orders of magnitude), and the sensitivity of this plasmon-based bioassay.

In an attempt to further increase the limit of detection and sensitivity, Rodriguez-Lorenzo et al developed plasmonic nanobiosensors and tested them for the detection of prostate specific antigen (PSA) as a model cancer biomarker [61]. The aim of these researchers was to overcome the limitation that conventional transducers generate signals that are directly proportional to the concentration of the analyte, which impedes the detection of ultra-low concentrations of analyte with high confidence. Rodriguez-Lorenzo et al improved the LSPR technology by programming crystal growth to favor either the formation of a silver coating around the transducer or the nucleation of silver nanocrystals in solution through the use of an enzyme that controls the rate of nucleation of silver nanocrystals on the plasmonic transducers. In so doing, they increased the sensitivity and robustness for the detection of PSA, with a detection limit of 4 × 10⁻²⁰ M in whole serum.

Ranallo et al developed yet another type of sensor—one capable of one-step fluorescent detection of both monovalent and multivalent proteins [62]. They designed a stem-loop DNA scaffold that presents a small molecule, polypeptide or nucleic-acid recognition element on each of its two stem strands (figure 2). The binding of an Ab to its target molecule triggers an opening in the stem, thereby enhancing the emission of an attached fluorophore/quencher pair. This rapid and selective sensor activation was sufficiently sensitive to facilitate the detection of specific proteins in complex samples, such as in serum. The team reported low nanomolar detection limits and no detectable cross-reactivity for five bivalent proteins (including four Abs) and two monovalent proteins (including a Fab fragment). This type of modular nanoswitch may offer advantages over existing methods by virtue of the lack of reagents needed, the lack of washing steps and the reduced sensitivity to temperature and other environmental factors that alter catalysis. This approach exemplifies the integration of Abs as specific recognition elements and as inducers of conformational changes that generate measurable signals.

Rissin et al, too, used a fluorescence technology, this time to address the need to increase the limit of detection of biosensors. They developed a platform that enabled the detection of single protein molecules in blood samples at ultra-low concentrations [63]. To this end, they prepared microscopic beads coated with specific capture Abs designed to bind proteins. The bead-Ab complexes were then incubated with the target analyte and labeled with a tracer Ab conjugated to an enzymatic reporter capable of generating a fluorescent product. Isolation of the bead-immunocomplexes in femtoliter-volume reaction chambers that were designed to hold only one bead in each well enabled the detection of single protein molecules by fluorescence imaging. This unique approach, termed digital ELISA, demonstrated the ability to detect as few as ∼10–20 enzyme-labeled complexes in 100 μl of sample and, thus, provided an alternative to conventional ELISA for the detection of clinically relevant proteins in serum at low concentrations (<10⁻¹⁵ M).

A number of papers have reported nanobiosensors based on magnetic nanoparticles, which are a class of nanoparticles that can be manipulated by the influence of an external magnetic field. They are widely used in various applications, such as magnetic resonance imaging, targeted drug and gene delivery, tissue engineering and cell tracking [64]. They have also been extensively used in Ab-based nanobiosensors by virtue of their high sensitivity, fast response, and reliability. A promising strategy to increase their sensitivity is by signal amplification of the optical signal derived from an enzyme-mediated colorimetric immunoassay. In an application of such a strategy, Gao et al developed a novel reverse assay whereby the absence of a target triggers a change in the signal and the relationship between the target concentration and the signal change is inverse. This strategy generated a highly sensitive reverse colorimetric immunoassay with the ability to detect low concentrations of proteins [65]. The authors described an experimental setup to produce a reverse colorimetric immunoassay for the ultrasensitive detection of PSA, as a model protein, in biological fluids. This strategy exhibited a wide detection dynamic range of 0.05–20 ng ml⁻¹ towards PSA with a detection limit of 0.03 ng ml⁻¹.

During the past decade, significant advances have been made in the generation of well-ordered nanostructures. Initially, DNA was used as the molecular building block for engineering such nanostructures [66–68]. The design process of such nanostructures, also termed DNA origami, confers the ability to control the order and shape of the building blocks, affording a variety of complex and scalable DNA-based architectures. Today, the available DNA nanotechnology has expanded the milieu of designed nanostructures beyond RNA and DNA aptamers by integrating proteins or other ligands and thereby enabling the introduction of specific functionalities into these well-ordered DNA designs. In particular, the incorporation of Abs into such nanostructures enhances their stability and, more importantly, turns the nanostructures into functional entities with high specificity.

He et al, for example, used antigen-modified DNA arrays as templates to direct Abs to assemble into high-density nanoarrays [69]. The resulting Ab nanoarrays presented a well-defined tetragonal order with a repeating distance of ∼20 nm. An additional important property of the nanoarrays was that all the Ab molecules had the same orientation, which was important for controlling their biochemical activities. Since the Ab nanoarrays are assembled in solution rather on solid substrates, they may be applied in both in vitro and in vivo studies, including single molecule studies of antigen–Ab interactions.

Rosier et al described a modular strategy to covalently conjugate oligonucleotides to proteins containing the Ab Fc domain by using a versatile photoreactive protein G adapter [70]. This modular approach enabled the decoration of DNA nanostructures with complex native proteins while maintaining their innate binding affinity. The main idea underlying this strategy was to utilize the specific interaction between the Fc portion of the Ab and protein G. By generating precise arrays with various proteins fused to the Ab Fc, the researchers demonstrated the potential versatility of the approach, which allows precise control over the nanoscale spatial organization of such proteins for in vitro and in vivo biomedical applications. In contrast to many DNA–protein conjugation methods, which require challenging chemical modifications to the protein of interest, the suggested method can be applied to a multitude of commercially available proteins, including Abs, growth factors, cell receptors, and cytokines.

In a different approach, Lee et al demonstrated how well-defined Ab-based nanostructures could be combined in diagnostics applications. They demonstrated the fabrication of sensitive 3D immunoassay platforms by the immobilization of IgG on the B domain of staphylococcal protein A (SPA_B) that was genetically inserted on the surface of a proteinticle [71] (where 'proteinticle is a nanoscale protein particle that is self-assembled inside cells with a constant 3D structure and surface topology' [71]). This methodology enabled control over the orientation of the variable domains of the bound IgG, thereby enabling effective capture of antigens and hence the engineering of a highly sensitive 3D IgG probe. The team used five types of proteinticles of different origins that differed in size, shape, and surface structure for the surface display of SPA_B. They showed that the dissociation constant K_D for the binding of IgG to SPA on the proteinticle surface was 1–3 orders of magnitude lower than the previously reported K_D [72], indicating more efficient and stable immobilization of well-oriented IgG Ab on the proteinticle surface. This study thus demonstrated how to genetically modify proteinticles—as nanostructure scaffolds—to fabricate sensitive and stable 3D IgG probes.

Wang et al developed a DNA-Ab nanostructure specifically for the detection of Streptococcus pneumoniae [73]. They designed an electrochemical immunosensor for the rapid detection of pneumococcal surface protein A (PspA) and an S. pneumoniae lysate from synthetic and actual human samples. Their immunosensor was composed of a DNA tetrahedron with a hollow structure, which was anchored on gold electrodes. The DNA tetrahedron nanostructure enabled the immobilization of the capture Ab at the correct orientation at which the binding site was turned towards the analyte. The composite nanostructures displayed excellent electrochemical activity toward PspA and exhibited good sensing performance toward an S. pneumoniae lysate. In addition, by virtue of the high Ab specificity, the immunosensor could specifically recognize and detect the PspA peptide both when mixed with other physiologically relevant components, such as BSA and lipopolysaccharide, and also when present in uncultured samples from the nasal cavity, mouth, and axilla of a human subject. This immunosensor could thus rapidly detect the presence of S. pneumoniae and thereby assist in decreasing the morbidity and mortality of the life-threatening diseases inflicted by this species of bacterium.

Nanostructures can also be leveraged for the detection of larger biological entities, such as whole cells. Khosravi et al developed a unique nanotube-Ab microarray for the rapid capture of breast cancer cells spiked in blood, without the necessity for pre-labeling, pre-fixation, or any other processing steps [74]. Here, too, the researchers relied on the high specificity of the incorporated Ab and reported a 55%–100% (mean 62%) capture yield of CTCs. One of the advantages of this assay is its ability to isolate viable cells, whereas magnetic-bead-based approaches can isolate only fixed, nonviable cells. With their capability for fast and sensitive cell capture, these nanotube microarrays have the potential to become useful tools for the capture and sorting of CTCs and will also find applicability in studies devoted to drug screening and intracellular signaling.

Yoon et al demonstrated another effective approach—based on functionalized graphene oxide nanosheets—to isolate CTCs from blood samples of patients with pancreatic, breast or lung cancer [75]. Functionalized graphene oxide nanosheets were adsorbed onto a patterned gold surface and chemically functionalized with Abs that target epithelial cell adhesion molecules. Even at a low concentration of target cells, CTCs were captured with high sensitivity exceeding that of other—Ab independent—approaches. With their novel approach, the team was able to isolate, capture, identify and characterize rare CTCs in the blood of cancer patients, which is highly desirable for early cancer detection.

Since Abs are extremely specific to their targets, it has been suggested that they be used as 'guided missiles' to increase the specificity of drug delivery to the target site while minimizing damage to healthy tissue. In the cancer scenario, target-specific Abs are used to functionalize nanoparticles as the vehicle for drug delivery. The functionalized nanoparticles (1–100 nm) can passively target the drug payload (high dose of a cytotoxic drug) to the tumor site, thereby minimizing systemic exposure and its associated side effects. The circulation half-life of the nanoparticles encapsulating any particular drug is dependent on their size, charge, composition and surface properties [76]. Nanoparticles can be classified according to their material composition, i.e. metallic, synthetic, liposomal and/or polymeric. Polymeric nanoparticles, which are based on natural polymers, such as dextran, alginate, guar and methylcellulose, offer some advantages over other nanoparticles, as they are more stable and allow facile triggering of drug release in vivo [77]. The specificity of nanoparticle delivery (true for all nanoparticles) to the target site and hence the therapeutic efficacy can be enhanced by the incorporation of mAbs or mAb fragments (figure 1(B)), which can be engineered to target specific biomarkers that are present on the target cell. Ab fragments are often preferred over whole Abs, since they are smaller and do not trigger complement activation. For conjugation to the nanoparticle, a suitable functional group has to be introduced onto the nanoparticle surface or the Ab. Several reactive groups—amino, carboxy, thiol and hydroxy—on the amino acids comprising the Abs can be used for conjugation.

Numerous nanoparticle-based drugs are currently in different stages of clinical trials. Phase I clinical trials of C225-ILs-dox, doxorubicin-loaded immunoliposomes functionalized with the Fab fragment of cetuximab (approved mAb that targets the epidermal growth factor receptor (EGFR)) have been completed, and phase II trials have been initiated [78]. EGFR can also be targeted by bispecific Abs conjugated to bacterially derived 'minicell' nanoparticles [79]. In some cases, the mAb itself is the therapeutic agent; for example, trastuzumab (Herceptin) is a therapeutic Ab that targets the HER2 receptor and hampers tumor growth through inhibition of downstream signaling and activation of immune mechanisms, such as Ab-dependent cellular cytotoxicity (ADCC). One of the drawbacks of this therapeutic agent is that it is rapidly internalized [80], and therefore several conjugated formulations have been developed to extend its circulation time, e.g. trastuzumab-emtansine (T-DM1) [81] and nanoparticle-based formulations [72]. Another problem that is often encountered in chemotherapy—the low solubility of certain drugs—may also be addressed by the use of Ab-functionalized nanoparticles as the delivery system. For this purpose, biopolymer particles were constructed by attachment of the targeting protein or Ab to an assembled micelle [82]. Micelle-attached whole Abs and Ab fragments were then evaluated for the enhanced delivery of poorly soluble drugs to various pathological sites in the body; they were found to be effective in recognizing various cancer cells in vitro and showed an increased accumulation in experimental tumors in mice compared with non-targeted micelles. For example, Torchilin et al developed a taxol-encapsulating micelle conjugated to anti-nucleosome 2C5 Ab. This formulation exhibited in vitro binding to various cancers and in vivo binding to Lewis lung carcinoma cells, with increased accumulation of taxol in the tumor and hence enhanced tumor inhibition [83]. DOX-PLGA-PEG micelles conjugated to anti-hepatocellular carcinoma F(ab')₂ were shown to enhance suppression of tumor growth in vivo compared with doxorubicin or DOX-PLGA-PEG micelles [84]. Qian et al used the anti-CD44 scFv Ab fragment to enhance the delivery of arsenic-containing micelles and showed inhibited tumor growth in vivo after treatment with these immune-micelles [85].

In a different type of study, Copp et al used cell membrane-coated nanoparticles to clear pathological Abs with the aim to minimize the disease burden without drug-based immune suppression [86]. The team used intact red blood cell (RBC) membranes stabilized by biodegradable polymeric nanoparticle cores to serve as an alternative target for pathological Abs in an Ab-induced anemia disease model. They demonstrated, in both in vitro and in vivo studies, the efficacy of RBC membrane-cloaked nanoparticles to bind and neutralize anti-RBC polyclonal IgG and, therefore, to prevent damage to healthy RBCs.

Nanaware-Kharade et al customized the in vivo behavior of an anti-methamphetamine (METH) single chain Ab with the aim to address the need for the development of effective medications for the treatment of METH abuse [87]. They developed a novel multivalent METH-binding nanomedicine by conjugating multiple anti-METH scFvs to dendrimer nanoparticles, via a polyethylene glycol (PEG) linker, to produce conjugates termed dendribodies. The team showed that nanoparticle conjugation did not impair the high affinity (K_D = 6.2 nM) and specificity of the scFv to METH. Such a dendribody platform could be used for preparing multivalent medications and could also be implemented to increase the serum residence time of other biological therapies for the treatment of numerous diseases.

Chen et al used gold nanoparticles conjugated to Ab fragments (anti-EGFR) as probes in the targeting of tumor cells [88]. Exposure of the nanoparticles to a green laser (λ = 532 nm), resulted in the production of sufficient thermal energy to kill urothelial carcinomas both in vitro and in vivo. The team reported that nanoparticles conjugated with Ab fragments were capable of damaging cancer cells even at relatively very low energy levels, while non-conjugated nanoparticles required energy levels 3 times as high under the same conditions. This energy differential facilitated the in vivo elimination of cancerous cells while preserving normal cells. After the treatment, the assembled nanoparticles can be removed to reduce their unwanted deposition in the body.

Ning et al produced SN-38-loaded nanoparticles conjugated with anti-CD133 Ab in attempt to improve the efficacy of chemotherapy with SN-38, a topoisomerase inhibitor used as a cytotoxic agent [89]. CD133 is a marker for colorectal cancer and has been proposed as a therapeutic target. Cells that overexpress the CD133 glycoprotein have been shown to exhibit enhanced tumor-initiating ability and tumor relapse probability. The team reported that their Ab-conjugated SN-38-loaded nanoparticles bound efficiently to HCT116 cells, which overexpress CD133, with excellent biocompatibility and strong CD133 binding affinity. They also showed that the cytotoxic effect of the Ab-coated nanoparticles was greater than that of non-targeted nanoparticles in HCT116 cells. These nanoparticles could target cells overexpressing CD133 and inhibit colony formation. Furthermore, in vivo studies in an HCT116 xenograft model demonstrated suppressed tumor growth and retarded recurrence after treatment with the Ab-coated nanoparticles. The authors concluded that this delivery system could eliminate CD133+ cells and could therefore be considered as a potential colorectal cancer targeted therapy.

Ramishetti et al described a novel strategy to specifically deliver small interfering RNAs (siRNAs) to murine CD4+ T cells by using targeted lipid nanoparticles [90]. The targeted lipid nanoparticles were surface-functionalized with anti-CD4 mAbs to facilitate delivery of the siRNA specifically to CD4+ T cells. Using this approach, the authors demonstrated how targeted lipid nanoparticles were bound and efficiently taken up into CD4+ T cells at several anatomical sites [90]. The approach was further developed to target mantle cell lymphoma (MCL) cells and induce cell-specific therapeutic gene silencing in vivo [91]. The latter studies [90, 91] demonstrated the potential versatility of Ab-based nanotechnology applications by altering capture Ab specificities.

In a more recent study, the delivery of modified messenger RNA (mmRNA) was achieved by loading lipid nanoparticles (LNPs) attached to a mAb with the aim to release the mmRNA into specific cells that would, in turn, manipulate gene expression. However, cell-specific targeting of LNPs is challenging due to a lack of selectivity. To address this challenge, a modular targeting platform named Anchored Secondary scFv Enabling Targeting (ASSET) was developed in which LNPs were coated with mAbs, enabling flexible switching between different targeting mAbs in accordance with the chosen target cell. With this approach, it is possible to construct various targeted carriers that deliver RNA molecules efficiently to various leukocyte subsets in vivo [92]. It was also shown that the ASSET technology could be exploited to deliver mmRNA specifically to inflammatory leukocytes. Such a study demonstrated a significant therapeutic effect in a mouse model of inflammatory bowel disease by manipulating the expression of interleukin 10 (IL-10) in Ly6c+ inflammatory leukocytes [93].

Nanomachines are nanoscale mechanical devices—nanorobots—that enable conversion of energy (whether light, electromagnetic, acoustic, or chemical) into precise mechanical motions. For many years, the realization of the potential of such devices seemed extremely challenging, but rapid advances in nanoscience and technology have recently led to the development of numerous novel applications of nanoscale mechanical devices. There are three main types of nanomachine. The first type, molecular machines, mostly several to tens of nanometers in size, can be produced from naturally existing biomolecules or synthetic biochemicals [94, 95]. The second type of nanomachine is produced from inorganic nanoparticles ranging in size from hundreds of nanometers to a few micrometers. They are mainly used in a highly controllable manner to perform various forms of mechanical actions, e.g. to transport, rotate, roll, drill, etc [96–99]. The third type comprises bioinorganic hybrid nanomachines. In these nanomachines, the biocomponent portion of the device serves as a microscopic engine that drives the inorganic components to generate reproducible motions. These devices range from a few micrometers to a few millimeters in size.

In recent years, a multitude of nanomachines have been developed to respond to specific molecular inputs. However, only a few have used Abs as active components to generate regulatory inputs. Ranallo et al addressed the challenge of generating regulatory inputs by designing a new class of DNA-based nanomachines that could load and release a molecular cargo upon the binding of a specific Ab to its target [100]. A DNA strand labelled with two antigens was loaded with a nucleic acid strand (see blue component in upper left pane figure 2) through a clamp-like triplex-forming mechanism. The binding of a bivalent macromolecule (here an antibody) to the two antigens caused a conformational change that reduced the stability of the triplex complex, with the consequent release of the loaded strand. The principle of these nanomachines was based on the conformational change that occurs in the interaction between an Ab and its target antigen, which induces the release of the active biomolecule. Such systems are highly versatile and, in principle, can be adapted to any Ab for which an antigen can be attached to a DNA-anchoring strand. Ranallo et al demonstrated that this approach could be extended to three different Abs and that the effect was specific and selective, even in complex environments. In addition, this type of nanomachine could reversibly load and release its cargo upon cyclic addition of the specific Ab and of the free antigen into a solution containing both the nanomachine and the cargo. Since Abs can target a wide range of proteins, Ab-powered DNA nanomachines may be useful in a wide range of applications, including diagnostics, controlled drug-release and in vivo imaging.

Douglas et al designed a robotic DNA device capable both of selectively interfacing with cells to deliver signaling molecules to cell surfaces and of reconfiguring its structure for payload delivery [101]. This nanomachine was controlled by an aptamer-encoded logic gate, enabling it to respond to a wide array of cues. Such nanorobots were loaded with combinations of Ab fragments and were used in two different types of cell-signaling stimulation in tissue culture. The authors demonstrated that biologically active payloads may be bound indirectly via interactions with Ab fragments. This may facilitate applications in which the robot carries out a scavenging task before targeted payload delivery.