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Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 National Science Review 5: 740–755, 2018 REVIEW doi: 10.1093/nsr/nwx134 Advance access publication 10 February 2018 CHEMISTRY Biomacromolecular nanostructures-based interfacial engineering: from precise assembly to precision biosensing 1,3 1 1,∗ 2,∗ Fan Yang , Xiaolei Zuo , Chunhai Fan and Xian-En Zhang ABSTRACT Biosensors are a type of important biodevice that integrate biological recognition elements, such as enzyme, antibody and DNA, and physical or chemical transducers, which have revolutionized clinical diagnosis especially under the context of point-of-care tests. Since the performance of a biosensor depends largely on the bio–solid interface, design and engineering of the interface play a pivotal role in developing quality Division of Physical biosensors. Along this line, a number of strategies have been developed to improve the homogeneity of the Biology & Bioimaging interface or the precision in regulating the interactions between biomolecules and the interface. Especially, Center, Shanghai intense efforts have been devoted to controlling the surface chemistry, orientation of immobilization, Synchrotron Radiation molecular conformation and packing density of surface-confined biomolecular probes (proteins and nucleic Facility, Shanghai acids). By finely tuning these surface properties, through either gene manipulation or self-assembly, one Institute of Applied may reduce the heterogeneity of self-assembled monolayers, increase the accessibility of target molecules Physics, Chinese and decrease the binding energy barrier to realize high sensitivity and specificity. In this review, we Academy of Sciences, summarize recent progress in interfacial engineering of biosensors with particular focus on the use of Shanghai 201800, protein and DNA nanostructures. These biomacromolecular nanostructures with atomistic precision lead China; National Key to highly regulated interfacial assemblies at the nanoscale. We further describe the potential use of the Laboratory of Biomacromolecules, high-performance biosensors for precision diagnostics. CAS Excellence Keywords: biosensor, interface engineering, homogeneity, orientation control, fused proteins, DNA Center for nanostructures Biomacromolecules, Institute of Biophysics, Chinese tion ; (ii) how does interfacial architecture affect INTRODUCTION Academy of Sciences, biomolecular adsorption, assembly, folding and dif- The biosensing interface between the solid–liquid Beijing 100101, China fusion [7,8]; and (iii) how do surface properties in- phases plays an important role for mass transfer, and School of fluence the folding free energy and structural dynam- Laboratory Medicine, electron mobility, energy exchange and signal trans- ics of biomolecules [9,10]? Hubei University of duction. By tailoring the interface with functional Along the lines to address these questions, a Chinese Medicine, molecules or materials, surface properties can be ef- number of strategies have been proposed to de- Wuhan 430065, China fectively modulated to ensure the homogeneity of velop high-performance biosensing interfaces with the biosensor surface and thus to improve biomolec- ∗ improved biomolecular recognition efficiency and Corresponding ular recognition, such as nucleic acid hybridiza- reduced non-specific adsorption [ 11]. Great efforts authors. E-mails: tion or antigen-antibody binding for efficient opti- have been made to increase the homogeneity and email@example.com; cal or electronic signal transduction [1–5]. Given firstname.lastname@example.org orderliness of interfacial assembly of enzyme pro- that this transduction process generally occurs at teins, antibodies or DNA probes . Especially, the solid–liquid interface, in-depth understanding rational interfacial design has proven to effectively Received 17 May of the interactions between biomolecules and the 2017; Revised 2 July realize the orientation control of the biorecogni- supporting interface is indispensable. For example, 2017; Accepted 3 tion molecules or reduce non-specific adsorption (i) what is the main difference between surface- July 2017 and avoid false positives. Among these approaches, confined and solution-phase biomolecular recogni- biomimetic interfacial engineering has shown great The Author(s) 2018. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. All rights reserved. For permissions, plea se e-mail: email@example.com Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 REVIEW Yang et al. 741 Head-on Tail-on Side-on Flat-on (b) (a) Binding site NH SS Random immobilization How? SS (c) Sugar Fc Antibody Oriented immobilization Figure 1. Structure and interfacial orientation of natural antibodies. (a) Schematic depiction of the structure of natural anti- body with two Fab and one Fc domain, as well as multiple reactive groups, such as amine, sugar and disulfide group. Antibody can be immobilized in either a random (b) with four typical orientations or oriented (c) fashion. promise for in-vitro biodetection . In nature, freedom for conformational change and functional biorecognition processes are generally highly effi- modulation. cient and specific, which are often based on synergic functions of ‘molecular machinaries’ that comprise multiple biomolecules in molecule-crowding intra- STATIC ENGINEERING BY CONTROLLED cellular settings. Construction of biomacromolecu- lar nanostructures represents a promising means to IMMOBILIZAITON OF PROTEINS mimic their intracellular counterpart and engineer Orientation control of proteins at the interface is biosensing interfaces in a programmable way . highly required to retain their bioactivities. How- In this review, we aim to summarize recent progress ever, proteins often adopt random orientations at in using biomacromolecule nanostructures for inter- the biosensing interface due to their multi-site in- facial engineering of biosensors primarily on gold teractions with the substrate. For example, Im- surfaces and with particular focus on structured pro- munoglobulin Gs (IgGs) (Fig. 1a) consist of one tein and DNA probes. Biosensors based on other constant fragment (Fc) and two variable antigen- surfaces such as graphene oxide, metal oxides and 2D binding fragments (Fab), resulting in random ori- transition-metal dichalogenides nanomaterials have entations of interfacial proteins: head-on (both Fab also been explored and could be found in several ex- on surface), tail-on (Fc on surface), side-on (Fc and cellent reviews [15–18]. We also provide perspec- one Fab on surface) or flat-on (all fragments on sur- tives on their applications in precision diagnosis. face) (Fig. 1b). To achieve optimal antigen-binding activity, antibodies should be immobilized with fa- vorable orientation of tail-on (Fig. 1c). Efforts to ad- dress this problem have been focused on precisely PROTEIN-BASED INTERFACIAL defined protein adorsption (non-covalent) and an- ENGINEERING choring strategies (covalent) . Bioactivities of protein molecules (i.e. antibodies Intermediate proteins (e.g. protein A or protein and enzymes) are responsible for high efficient anti- G) (Fig. 2a) with multiple binding domains specific gen recognition or direct electron transfer at the sen- to the Fc part of the antibody were employed to im- sor interface. Nevertheless, it is challenging to retain mobilize the antibody with favorable tail-on orien- their natural bioactivities and conformations when tation. As compared to their randomly immobilized proteins are immobilized on the sensing interface via counterparts, the oriented antibodies improved physical adsorption or covalent conjugation . the biosensing sensitivity and specificity [ 22,23]. Since proteins can interact with the interface via However, even partially oriented intermediate pro- their various surface groups, they tend to adsorb on teins may induce random orientations of antibod- the interface with random orientations that often do ies, which still poses a limit. To further improve not favor their bioactivities . Moreover, surface the protein orientation, a highly organized IgG- attachment might also alter the native conformation protein with 3D structures and exposed antigen- of proteins , thereby degrading their functions. binding sites was designed . However, this ori- Hence, a range of static and dynamic interfacial engi- ented immobilization strategy is heavily dependent neering approaches have been proposed to assemble on solution pH that negatively affects the protein proteins on the interface with better orientation and G orientation on gold and subsequent antibody Fab Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 Streptavidin 742 Natl Sci Rev, 2018, Vol. 5, No. 5 REVIEW (a) (b) Antibody Enzyme Protein A/G Linker Sugar Cys Non-covalent Covalent orientation orientation (c) Antibody DNA Antigen Antigen Cell Cell DNA DNA Cell/Antigen/DNA (d) Regenerable SBP-tag Reversible Antigen (e) MBP Ru(II) Maltose SAM Gold electrode Gold electrode Figure 2. Protein-based interface modulation. (a) Protein A/G mediated non-covalent orientation of antibodies. (b) Cova- lent orientation of antibodies by either reactive groups distributed on the surface of antibody or fusion protein technology. Adapted from . (c) Protein-DNA conjugates mediated orientation of antibodies. (d) Proteins reversibly modulate the sens- ing interface. Adapted from . (e) MBP dynamically modulates the electrochemical interface. Adapted from . Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 REVIEW Yang et al. 743 DNA-antibody conjugate renders the biosensing in- binding . Cysteine-functionalized protein G terface regenerable via facile water rinsing and ro- (Cys-protein G) has proven successful for con- trolled orientation of protein A or protein G at the bust to resist the denaturation induced by surface ef- interface . A recombinant Cys-protein G trimer fect. synthesized by repeated linking of protein G via flex- Scaffolded protein nanostructures provide a ible linkers can enhance the bioactivities of antibod- powerful means to oriented assemble proteins at ies immobilized on magnetic silica nanoparticles. the interface. For example, Zhang et al. fused a Compared to a Cys-protein G monomer probe, the coding sequence of streptavidin recognition peptide trimer improved the sensitivity by 10-fold. By geneti- (streptag) to a specific site (3 end) of the phoA cally fusing protein A domain to a Cys-exposing vari- gene that codes for E. coli alkaline phosphatase ant of Escherichia coli protein ompA, favorable bind- (EAP) . This fused protein was used for oriented ing orientation and antibody presentation were real- immobilization of EAP enzyme on microtiter plates ized . Similarly, a ‘super-oriented IgG’ was con- via streptag–streptavidin binding. With the insertion structed to attach to the oriented protein A at the in- of a flexible linker peptide coding sequence between terface via enzyme conjugation, leading to enhanced the streptag and EAP sequences, the enzyme activity affinity up to ∼100 fold over the partially oriented of immobilized EAP increased by ∼8.4-fold over IgG physical immobilized at interface . the fused protein without linker sequence. The linker peptide provides a spacer that minimizes the Although the interfacial orientation control with steric hindrance between EAP and streptavidin, intermediate proteins can enhance biosensing per- enhancing the orientation effect. In a further step, formance, this non-covalent interaction between in- by incorporating a linker peptide with a cysteine termediate protein and interface is generally weak residue at the C-terminal of glucose oxidase (GOx), and sensitive to environmental changes, such as pH, they engineered a fusion structure (GOx-linker- ionic strength and temperature . In response, cysteine) that can be immobilized on gold surfaces direct covalent immobilization of proteins provides a feasible way to engineer the biosensing inter- with Au–S bond or on a silanized glass surface face. Classic reactions of inherent reactive groups via disulfide bond (Fig. 2b) . Importantly, this and tailor-made modifications on protein molecules fused cysteine enables the GOx to be immobilized have been used to improve the orientation and ho- at the interface with well-controlled orientation, mogeneity of proteins at the interface (Fig. 2b). thus forming a homogeneous biosensing interface. Amine (NH ) groups in the lysine side-chain on the With the synergistic effect of the linker spacer and antibody surface are usually used for relatively ran- interfacial homogeneity, a higher and more stable dom covalent immobilization, although it generally electrochemical current response was obtained as lacks orientation control [29,30]. To improve the compared to the GOx without fused cysteine and orientation control, in contrast, the unique carbo- linker spacer. hydrate moiety at the Fc portion of an antibody is Although the covalent orientation of proteins is more amenable to orient proteins via covalent im- effective, the high-affinity interaction (e.g. biotin– mobilization. In the presence of periodate sodium streptavidin binding) is usually irreversible, which  or boronic acid , the carbohydrate vici- makes the biotin–streptavidin based assembly non- nal hydroxyl groups would be oxidized to aldehy- regenerable. To realize a regenerable biosensing interface (e.g. surface plasmon resonance, SPR), two des and cyclic boronate esters, respectively, which streptavidin affinity tags, nano-tag and streptavidin- provide oriented covalent antibody immobilization binding peptide (SBP-tag), were employed (Fig. 2b). Analogously, the disulfide group in the (Fig. 2d) . Both of them can specifically interact hinge region of antibody was also exploited for ori- with streptavidin while the binding affinity (K ented immobilization of antibody via reduction into ∼4-17 nM) is weaker compared to the biotin– thiol groups (Fig. 2b) . These methods of cova- streptavidin binding (K ∼1 fM), thus enabling lent coupling of antibody at the interface has proven widely useful for antibodies with multiple activatable the easily controlled association and dissociation. sites on antibodies. With this tunable binding mechanism, Zhang and In addition, DNA can also be used to immobi- coworkers developed a SPR biosensor chip coated lize antibody, serving as both spacer and binding do- with streptavidin for reversible, site-directed protein main. Heath et al.  established a DNA-encoded immobilization mediated by the nano-tag . The antibody library for spatially multiplexed detection streptavidin surface could be regenerated repeatedly of nucleic acids, proteins and cells (Fig. 2c). Impor- without loss of activity even by injection of 50 mM tantly, the hybridized duplex probe is rigid enough to of NaOH solution. This reversible biosensing maintain a favorable orientation (upright) of the an- interface could be readily generalized to build other tibody at the interface . More importantly, this SPR biosensors, permitting anchoring of various Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 744 Natl Sci Rev, 2018, Vol. 5, No. 5 REVIEW proteins on the streptavidin surface in a stable, related natural or designed MBP-like proteins that site-directed and reversible fashion. would undergo a similar ligand-induced conforma- tional change . Allosteric protein switches are ubiquitous in the biological signal transduction system, which en- DYNAMIC ENGINEERING BY able cells to sense and respond to specific molec- CONFORMATIONAL CHANGE OF ular events. Inspired by nature, tailor-engineering PROTEINS of protein switches with custom input and out- In nature, biomolecular recognition is generally ac- put functions is significant in molecular diagnostics companied by conformational changes of proteins, and physiological decoding . To demonstrate such as ligand-induced protein folding or unfolding. this concept, RG13, a fusion protein of MBP and Such a special conformation-switchable mechanism TEM1β-lactamase, was engineered as a model pro- can be used to engineer the biosensing interface in tein switch with electrochemically activated switch- a dynamic manner . It can also be extended to ing behavior by reducing the disulfide bonds in the design a biosensor exclusively based on the confor- switches . In this approach, an electrochemical mational switch of proteins . Nevertheless, most signal can be used as an exogenous input to control proteins, unlike single-stranded (ss)DNA, do not the on/off state of protein switches by modulating the oxidation state of an introduced disulfide bond undergo significant conformational changes upon on the electrode surface. The presence of maltose is ligand binding. Hence, a highly sensitive method the key to activating the enzyme activity due to the that can precisely probe the weak conformational al- induced large hinge-bending conformational change terations is desirable. Extensive studies have shown in the MBP domain of RG13. This strategy allows the that electrochemical sensors are capable of trac- allosteric protein switch to dynamically regulate the ing the relatively small conformational changes of interface signaling. a surface-confined, redox-labeled macromolecule. Although the protein switch or folding is ef- Since heterogeneous electron transfer between an fective to engineer the biosensing interface, the electrode and a surface-confined redox molecule ex- ligand-induced conformational change in protein hibits an exponential dependence on both distance switches is relatively small. In contrast, a more and the Marcus coupling factor , even small con- flexible peptide, like aptamer, can trigger a large formational changes in proteins can produce large structure change by binding to its corresponding an- variations in electron-transfer rates, which in turn translate into measurable changes in electrochemi- tibody. Lai and coworkers  reported an electro- cal signals. For example, Benson et al.  exploited chemical peptide-based dynamic biosensor for the the ligand-induced hinge-bending motions in the sensitive and specific detection of HIV anti-p24 an- electrode surface-confined maltose binding protein tibodies where a highly antigenic epitope from the (MBP) that they used to engineer the biosensing HIV-1 capsid protein, p24, was used as the recog- interface in a dynamic fashion (Fig. 2e). In this re- nition peptide by modifying with MB (methylene port, a gold electrode was first assembled with a func- blue) and thiol at two ends, respectively. Of note, tional monolayer, serving as a binding interface for this epitope is a short linear peptide lacking defined site-specific immobilization of proteins [ 40]. Subse- secondary structure, and adopts predominantly an α-helical conformation in native state. Thus, the quently, the MBP protein was immobilized on the binding of the target antibody facilitates the forma- electrode surface with a specific orientation such that tion of a more rigid complex that induces a large a redox reporter group (ruthenium complex) was conformation change. This dynamic peptide-based fixed on the electrode for electrochemical readout. biosensor achieved a detection limit of 10 nM, and As the target ligand of maltose binds, the target- had a dynamic range that is broader than the typical induced hinge-bending motion in the protein pro- concentration range commonly observed with HIV- ceeds, which moves the Ru(II) reporter away from infected patients. the electrode. This target-responsive dynamic mod- ulator in turn triggers a concentration-dependent decrease in the observed electrochemical response, DNA-BASED INTERFACIAL ENGINEERING which provides the electronic detection of maltose at mM concentrations. It should be noted that the rel- Single-stranded (ss-)DNA can hybridize to its com- atively low sensitivity (at the mM level) of this dy- plementary DNA strictly obeying the Watson-Crick namic sensor is limited by the low affinity of MBP base-pairing rules, which allows DNA recognition against maltose (K = 4mM) butdoesnotpose a and structural assembly in a highly predictable fundamental restriction of this strategy. Apparently, and finely programmable manner. These advan- this method can be generalized to detection of other tages open opportunities for biosensing interfacial Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 REVIEW Yang et al. 745 d d (b) (a) Flat-on (c) (d) Dense Diluents Figure 3. ssDNA-based interface modulation. (a) Schematic depiction of an ideal DNA recognition layer with a large inter- molecular distance and linear upright orientation. (b) Sparse probes are prone to lying flat-on the surface due to non-specific adsorption. (c) Densely packed short DNA probes adopt an upright conformation, but yield poor hybridization efficiency be- cause of reduced accessibility. (d) Diluents, such as MCH and OEG, co-assembled with ssDNA to favor the upright orientation on the surface and reduce non-specific adsorptions. engineering. It was reported that the large inter- teractions exist between the DNA bases and the gold probe distances and upright orientation of surface- surface via multiple nitrogen atoms , which al- tethered DNA probes is imperative to realize lows ssDNA molecules to lie down on the Au sur- efficient hybridization. However, engineering an face, resulting in a largely limited accessibility of upright and accessible DNA recognition layer is the target sequences with reduced hybridization effi- challenging because of the unexpected surface ciency (Fig. 3b). Such non-specific adsorption onto adsorptions, disordered conformations and inho- the Au via Au–N interactions has been confirmed mogeneity of grafting density of DNA bioprobes at by Tarlov and coworkers . Further characteriza- the biosensing interface. Static and dynamic inter- tion, such as X-ray photoelectron spectroscopy and face engineering using DNA and DNA structures Fourier transform infrared spectroscopy, revealed have been developed to overcome this challenge. that the nonspecifically adsorbed ssDNA could not be removed, even with extensive rinsing or heating to 75 C, and the ‘specifically’ anchored ssDNA mono- layer is not oriented perpendicularly to the sur- STATIC ENGINEERING BY CONTROLLED face, especially at low densities. Short DNA probes IMMOBILIZATION OF DNA PROBES tended to orient parallel to the surface, whereas the relative long strands preferred to form disordered In a typical ssDNA probe-based DNA biosensor, film due probably to adjacent entanglement [ 48]. Al- an efficient probe–target hybridization process is though the densely packed short DNA probes may fundamental for improving the biosensing perfor- assume an upright conformation on the surface, the mance, particularly in sensitivity and specificity. restricted target accessibility would offset this advan- Nevertheless, high hybridization efficiency depends tage of favorable probe orientation (Fig. 3c). on a favorable orientation (upright) of DNA probes To help address this dilemma, Tarlov et al.  and rational inter-probe distance (Fig. 3a) . It is expected that the ssDNA probes can adopt an introduced a coassembling small molecule (mercap- upright orientation at the biosensing interface via a tohexanol, MCH), synergizing with SH-DNA to en- single-point attachment. By taking thiolated DNA gineer the recognition ability of bioprobes at the in- (SH-DNA) as an example, however, significant in- terface (Fig. 3d). This ‘helper’ molecule is able to Upright Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 746 Natl Sci Rev, 2018, Vol. 5, No. 5 REVIEW largely remove the nonspecifically adsorbed DNA sensitivity of biomolecular detection depends not and meanwhile protrudes the surface-attached DNA only on the affinity between biomolecules, but also probes into solution phase via the repulsion between on the interfacial properties of the biosensors , the net negative dipole of alcoholic terminus and the the size reduction of biosensors, particularly to the negatively charged DNA backbones. These mixed ss- nanoscale, usually accelerates the mass transport DNA/alkylthiol monolayers have been extensively rate and improves the sensitivity [67,68]. How- investigated by varied surface-measuring techniques ever, the limited space available in nanosensors and confirmed with a favorable upright orientation restricts the effective probe numbers and biorecog- of ssDNA bioprobes . This synergic static mod- nition events. To address this challenge of size re- ulation strategy was widely employed in engineering duction, a trans-scale biosensor that incorporates DNA biosensors and biochips [51–53]. However, nano-architectures into macroscopic surfaces is nec- the co-assembled small molecule diluents (MCH) essary . Nevertheless, reproducible engineering cannot resist the non-specific protein adsorption of nanostructured surfaces with well-defined topog- effectively. In response, a ssDNA/oligo-ethylene raphy remains technically difficult, though high-cost glycol (OEG) mixed monolayer was used to im- photolithography potentially offers a route to the prove the protein resistance of the biosensing inter- fabrication of nanostructures at the wafer-scale . face, particularly in complex matrices, such as blood Recently, Fan et al.  developed a conceptu- [54,55]. We also note that relatively rigid double- ally new ‘soft lithographic’ strategy to reproducibly stranded (ds) DNA molecules represent another engineer and programmably modulate a biosens- route to improving interfacial probe arrangement ing interface using well-defined 3D DNA nanos- with enhanced sensitivity and specificity, especially tructures (Fig. 4a). By patterning the macroscopic for toehold design . gold electrode with tetrahedral DNA nanostructures Although helix structures or the co-assembled (TDNs) varying in sizes, the detection limit of DNA diluents can aid DNA probes to adopt a favorable sensors can be programmably tuned over four orders orientation at the interface to some extent, the probe of magnitude. density is still a critical factor controlling the kinet- A typical DNA tetrahedron-structured probe ics of target/probe hybridization . Interestingly, (TSP) was self-assembled with a relatively long, a simple optimization of the assembly concentration probe-bearing oligonucleotide and three thiolated of DNA probes can accurately modulate the inter- oligonucleotides, which carries a pendant DNA facial probe average density. One concern in this probe (hybridizable domain) at one vertex and approach is that local lateral interactions inevitably three thiol groups (-SH) at the other three ver- exist in DNA films, particularly for long sequences tices (Fig. 4b), allowing it to firmly anchor on the [58,59]. This enables the prediction of the most fa- gold surface via Au–S bonds . Of note, this 3D- vorable hybridization in the ‘Langmuir’ (L) regime structured probe demonstrates several advantages hard to reach, because it only exists in the limits of over its single-stranded or duplex counterparts in the sparse films where probes are so far apart that they engineering of biosensing interfaces. First, the high do not interact with each other . From a single- mechanical rigidity of TSP allows it to adopt a highly molecule view , the aggregation patches on the ordered, upright orientation at the Au surface, which Au surface can significantly reduce target accessibil- was stabilized by the three thiol ‘legs’ that greatly ity, which, nevertheless, could not be easily elimi- increase the stability of the surface-confined probes nated using MCH as the dilution molecule. To bet- by ∼5000 times as compared to the mono-thiolated ter address this challenge, these empirically ‘static’ DNA strands . Second, the TSP structures avoid modulation methods dependent on the scheduled inter-probe entanglement (in ssDNA probes) via probe/diluent ratio may need to couple with nanos- spatially segregated pendant probes, and also re- tructured surfaces or conceptually new probe-design duce the surface effects by positioning the probes strategies. in solution-phase-like settings [ 72]. Importantly, it DNA nanostructures can be used to construct is able to precisely anchor a single DNA probe on a scaffolded biosensing interface to tune the sen- a single TDN, which is hard to achieve with inor- sitivity of biosensors in a programmable fashion ganic nanostructures. Third, the TSP-decorated sur- [14,62]. So far, researchers have engineered a va- faces are protein-resistant, and the ‘helper’ molecule, riety of DNA nanostructures with well-defined MCH, is not necessary in this case, both of which al- dimension, topography and precisely controlled low the TSP-based sensors to be deployed directly functions by using DNA nanotechnology. These in complex matrices, such as serum, for practical ap- functional DNA nanostructures have been actively plications. In addition, diffusion and convection at exploited to develop in-vivo or in-vitro biocomput- the nanostructured interfaces are also expected to be ing and biosensing devices [63–65]. Because the higher than those at macroscopic ones . Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 REVIEW Yang et al. 747 Figure 4. DNA nanostructure-based programmable modulation on the sensing interface. (a) Assembling DNA nanostructures with varying sizes for programmable modulation on the sensing interface . (b) A universal biosensing platform based on tetrahedron-structured DNA probes (TSPs) . Reprinted with permission from [14,70]. Copyright 2015 and 2010 by WILEY-VCH, Weinheim, respectively. (c) TSPs-based E-DNA sensor for microRNA detection . (d) TSP-conjugated antibody for sensitive PSA detection amplified with HRP-AuNP [ 79]. (e) TSP-conjugated aptamer for sensitive exo- some detection . (f) TSP-conjugated aptamer-HCR for sensitive detection of cancer cells . (g) TSP-mediated capillary microarray for multiplexed bioassays, achieving detection limits of 1 μM and 0.1 nM for small molecules (ATP and cocaine), respectively . Reprinted with permission from [79,82,83,93]. Copyright 2014 and 2017 by the American Chemical Society. Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 748 Natl Sci Rev, 2018, Vol. 5, No. 5 REVIEW In TSP-based sensors, the lateral distance be- scaffold to build immuno-sensing layers by conju- tween probes is dominated by the TDN scaffold, gating with antibodies. For example, antibodies of and thus this distance can be precisely tuned at the tumor-necrosis-factor alpha (TNF-a) can be eas- nanometer scale by varying the size of TDNs . ily and reversibly anchored onto DNA nanostruc- To this end, five types of TDNs (TDN-7, TDN- tured surfaces via a DNA-bridged antibody link- 13, TDN-17, TDN-26 and TDN-37) with different ing with EDC-NHS chemistry, which converts the sizes have been rationally designed . The num- DNA recognition layer into a protein recognition bers (7, 13, 17, 26 and 37) designate the base pairs one . Next, Zuo et al.  reported another of each edge in the TDNs. Because each base pair antibody immobilization strategy that uses highly was separated by 0.34 nm in a double helix, the edge efficient click chemistry instead of a carbodiimide lengths of the TDNs were able to be calculated with reaction to conjugate the PSA antibody with a DNA- precision, equal to 2.4, 4.4, 5.8, 8.8 and 12.6 nm, re- bridged TDN scaffold (Fig. 4d).Bypreciselycon- spectively (Fig. 4a). It was observed that the elec- trolling the nanospacing of anchored antibodies trode surface density of TDN probes was inversely with a nanostructured scaffold, they achieved an ex- proportional to the size of the TDN, which was fur- tremely high sensitivity (1 pg/mL) in PSA detec- ther confirmed by statistical analysis that revealed tion. Instead, a more direct, DNA-bridge-free ap- the nearly linear relationship between the lateral dis- proach was introduced to anchor anti-IgG on the tance and TDN size. Such an ability to precisely TDN scaffold for ultrasensitive IgG and bacteria regulate the lateral distance can also tune the sur- quantification [ 81,86,87]. By replacing the pendant face hybridization capabilities, including hybridiza- probe with an expanded nucleotide-containing ap- tion kinetics and efficiency, which depends heavily tamer, Tan et al.  developed a TSP-assisted ap- on the probe distance. By regulating the probe dis- tasensor for direct capture and detection of hepato- tance via the deployment of TDNs with varied size, cellular exosomes (Fig. 4e). This TSP-assisted ap- the detection limit of the biosensor was programmed tasensor demonstrated improved accessibility and . This result is consistent with that reported by detected the exosome with 100-fold higher sensi- the Howorka group , in which the molecular tivity as compared to the ssDNA-functionalized ap- recognition of receptors on TDN-scaffolded surface tasensor. In combination with a multibranched hy- was improved by an order of magnitude. bridization chain reaction that offers multiple biotins With the use of TDNs, a variety of ultrasensitive and branched arms, this TSP-functionalized inter- biodetections, such as nucleic acids [70,75–78], pro- face has been engineered to realize multivalent cap- teins [79–81], exosomes  and cells , have ture and highly sensitive detection of cancer cells been conducted. By using TSP-patterned gold elec- (Fig. 4f) . trodes, Pei et al.  engineered a biosensor with The TSP-based biosensing interface has also a typical sandwich hybridization strategy, in which proven to be an effective platform for the detection the upright probe can selectively capture its com- of small molecules, ions and even multiplex targets, plementary sequence with high discrimination abil- because the suspended probe can be easily replaced ity towards non-cognate sequences (Fig. 4b). The with various functional nucleic acids. As a proof free domain was subsequently flanked by the bi- of concept, a split aptamer for a small molecule, otinylated reporter probe that induced the produc- cocaine, was incorporated into TDN to develop a tion of electrochemical signals via the substrate catal- performance-enhanced electrochemical sensor for ysis of horseradish peroxidase (HRP). In combi- cocaine . To realize higher sensitivity, another nation with hybridization chain reaction amplifica- electrochemical cocaine sensor was presented based tion, this tetrahedral platform improved the detec- on structure conversion from a frustum pyramid to tion limit to 100 aM . For microRNA detection, an equilateral triangle . In this approach, the a synergic TSP-based electrochemical sensor was presence of cocaine triggered the aptamer-bearing constructed by integrating with rolling circle ampli- DNA nanostructure change from ‘Close’ to ‘Open’, fication (RCA) and silver nanoparticles that were at- achieving a detection limit of 0.21 nM. Likewise, tached to the RCA products in tandem . This an anti-ATP aptamer was immobilized at the top TSP-RCA sensor achieved a limit of detection as of the tetrahedron, thereby forming an ATP sensor low as 50 aM. To further improve the sensitivity, that could detect a concentration as low as 0.2 nM Wen et al.  increased the inter-probe distance . In conjunction with mercury-specific oligonu- 2+ using interfacial engineering and multi-enzyme am- cleotides, Yin et al.  constructed a turn-on Hg plification, which yields an extremely low detec- sensor with a detection limit of 100 pM, which was tion limit of 10 aM (∼600 microRNA molecules 100-fold lower than that obtained with a ssDNA- in 100 μL) (Fig. 4c). In addition to direct use as a based sensor. As a versatile probe scaffold, TSP can capture probe, the TDN can also serve as a probe easily evolve into a microarray platform by replacing Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 REVIEW Yang et al. 749 the pendant probe with the corresponding target- one-step response strategy, Lubin et al. suc- responsive probes. Recently, Li and coworkers fabri- cessfully detected DNA authentication tags that cated a high-throughput addressable microarray by are associated with paper or drugs. Later, Lai and covalently coupling TSP onto glass substrates for coworkers  demonstrated a sequence-specific multiplexed analysis with improved sensitivity and detection of unpurified amplification products of specificity [ 92]. The detection limits of this sensor thegyrBgeneof Salmonella typhimurium, signifying array for miRNA (let-7a), PSA and small molecule a promising way towards rapid, sample-to-answer (cocaine) were 10 fM, 40 pg/mL and 100 nM, pathogen detection. respectively. To accelerate the target binding, Qu The hairpin structure of DNA bioprobes at the in- et al.  reported a rapid (<5 min) arrayed DNA- terface can be employed to sterically hinder the ac- nanostructure-supported aptamer pull-down (DNa- cessibility of enzymes to bioactive sites at one end Pull) assay under convective flux in a glass capillary of the hairpin DNA, and thus a target-responsive (Fig. 4g). This DNaPull assay allowed multiplexed ‘signal-on’ signaling would be generated. Liu et al. analysis of the contents in droplets with nano- or pi-  developed a highly sensitive enzyme-based E- coliter volumes, achieving detection limits of 1 μM, DNA sensor that pushed the detection limit down and 0.1 nM for small molecules (ATP and cocaine) to low femtomolar concentrations (Fig. 5b), nearly and a biomarker (thrombin), respectively. three orders of magnitude lower than that of the ini- tial E-DNA setup , where the sensitivity is lim- ited to picomolar concentrations, partially because one Fc label can only transfer one electron from/to DYNAMIC ENGINEERING BY the Au electrode. In this work, however, one HRP CONFORMATIONAL CHANGE OF DNA 4 can convert ∼10 reactions of hydrogen peroxide Dynamic engineering is probably more attractive to water; the target binding-induced signal change due mainly to the target-induced conformational could be greatly amplified. Wei et al.  devel- oped a similar sensor for electrochemical detection change. These conformation-switchable DNA of salivary mRNA targets with 0.4 fM sensitivity. This molecules, such as hairpin (quadruplex and pseu- hairpin capture probe can also be designed with an doknot with intramolecular secondary structures), external toehold and immobilized on the gold elec- aptamer and DNAzyme, possess relatively rigid and trode surface of quartz crystal microbalance (QCM) ordered structures that may, in principle, prevent . Through the toehold-mediated strand dis- inter-strand entanglement at the interface and/or increase the orderliness of DNA bioprobes. In placement reaction, this QCM biosensor achieved 2003, Heeger and coworkers [94,95] developed a highly selective and sensitive detection of single- a conceptually new electronic equivalent of the nucleotide polymorphism (SNP) in the p53 tumor molecular beacon, the electrochemical DNA suppressor gene. (E-DNA) sensor, which exploits target-induced In addition to typical hairpin DNA probes, it is conformational changes of hairpin (stem-loop) also possible to design other dynamic probes, such probes on Au electrode surfaces (Fig. 5a). This as partial duplex , pseudoknot , triple- surface-confined ‘dynamic’ probe was labeled with stem  and triplex structures , to engineer an electroactive ferrocene (Fc) at the 5 end and a biosensing interfaces. For example, a partially com- thiol at the 3 terminal. Initially, the hairpin localizes plementary duplex probe was designed for a signal- Fc proximal to the electrode surface, thus enabling on, label-free electronic DNA sensor that achieved a rapid electron transfer. After hybridization, the a sub-picomolar concentration detection limit with stem opened and extended to a rigid duplex, forcing a strand displacement mechanism (Fig. 5c) . Fc away from the electrode surface. This dynamic This improved detection limit is believed to be at- response allows separation between Fc and the tributed to the rapid shift from rigidity (duplex) to electrode surface reaching several nanometers that flexibility (ssDNA) of the sensing probe. Next, a spe- cial pseudoknot probe that consists of two stem- yields a large and measurable change in electro- loop structures sharing one strand as the stem or chemical signal because of the exponential decay loop was deployed directly in complex matrices, of electron transfer with distance at the interface. such as blood serum for highly sensitive DNA de- This sensor achieved a selective detection of 10 pM tection (Fig. 5d) . Furthermore, Xiao et al. target DNA featuring in single-step and electronic  used a triple-stem structured probe to per- (electrochemical) detection. More importantly, this design utilizes only the intrinsic conformational form SNP assay even in the complex medium of change of surface-confined DNA probes to modu- blood serum (Fig. 5e). A thermodynamic analysis late interface signaling, avoiding the introduction suggested that this triple-stem probe takes advan- of exogenous reagents. By using such a dynamic, tage of the large thermodynamic changes in enthalpy Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 750 Natl Sci Rev, 2018, Vol. 5, No. 5 REVIEW Figure 5. DNA-based dynamic interface modulation. (a) The stem-loop-structured probe design for a reagentless electrochemical DNA (E-DNA) sensor . (b) A signal-on stem-loop-structured E-DNA sensor with HRP-catalyzed signal amplification [ 98]. (c) Stand displacement reaction-based signal-on electronic detection . Reprinted with permission from [94,101]. Copyright 2003 and 2006 by National Academy of Sciences, USA. (d) Two-stem- loop-structured E-DNA sensor . (e) Three-stem-loop-structured E-DNA sensor . (f) A conformation-switchable triplex E-DNA sensor . 2+ (g) DNAzyme-based dynamic probe as an ion (Pb ) sensor . (h) Dynamic DNA aptamer as an ATP sensor . Reprinted with permission from [98,102–105,107]. Copyright 2007, 2008, 2009 and 2014 by the American Chemical Society. can bind with its complementary target sequence and entropy resulting from major conformational through two distinct hybridization modes, namely rearrangements in target-responsive binding, lead- ing to exquisite sensitivity to single-base mismatches Watson-Crick recognition and Hoogsteen interac- . Another highly specific E-DNA sensor was tion. These bindings lead to the formation of a triplex also developed by using a triplex probe (Fig. 5f) that can improve both the affinity and specificity of . In this approach, a clamp-like DNA probe molecular recognition. Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 REVIEW Yang et al. 751 Functional nucleic acids, such as DNAzymes and or DNA nanotechnology allows the design and en- aptamers (RNA or DNA), can also be used to dy- gineering of proteins with site-specific modification namically engineer the biosensing interface. A typi- and DNA nanostructures, respectively, which con- 2+ cal Pb -responsive DNAzyme probe complex was siderably improves the capacity to control the probe 2+ employed for electrochemical detection of Pb density, orientation, homogeneity and accessibility 2+ . In the presence of Pb , the rigid structure at the interface. Based on these interface engineer- of the complexes would be cleaved, which produced ing strategies, a variety of biosensors have been specific electrochemical signals. Similarly to ion- tailored to probe biochemical substances, antigen- dependent DNAzyme, aptamers change their con- antibody binding or nucleic acid hybridization for formations in response to ions (Fig. 5g). The pres- highly sensitive detection of health-related enzyme ence of K can induce a contractile DNA duplex substrates and molecular biomarkers, such as mi- probe (K aptamer), which incorporated two short croRNA, DNA and protein, as well as tumor-derived separated motifs of G/G mismatches, which tran- exosomes and cancer cells. Despite the progress, sev- sited from the duplex DNA to the G-quadruplex, eral formidable challenges exist. First, in protein- and thus changed the charge-conducting properties based interface engineering, the indirect use of pro- of the DNA structure . Beyond ions, aptamer tein A or G reduces the efficiency. Thus, a more can also specifically bind with small molecules and direct approach for anchoring oriented protein at biomacromolecules. Zuo et al.  designed an the interface should be explored in the future, such adenosine triphosphate (ATP) sensor using an anti- as direct coupling of the Fc portion of the anti- ATP aptamer (Fig. 5h). Their study verified that body with a rigid spacer (e.g. dsDNA) for cova- ATP could stabilize the tertiary aptamer structure lent immobilization, which permits proteins with fa- at the surface and could responsively denature the vorable spatial orientation and homogeneity in a initial aptamer duplex. By incorporating a DNA ap- rational density. Designing one-step conformation- tamer into the three-way junction elements, a ratio- switch protein with large distance change is another nally designed probe architecture for the detection way to enhance the target response of the protein and quantification of thrombin (a plasma protein) interface in future dynamic sensors . Second, was reported . in DNA-based interface engineering, the rigidity- Compared to static engineering, dynamic engi- and flexibility-tunable DNA probe is highly desir- neering has shown superior properties in several able in designing surface-confined single-step DNA aspects. First, the enhanced rigidity over the lin- sensors by controlling the upright orientation and ear probes offers better-ordered orientation of DNA conformation switch. DNA tetrahedron is a versa- probes at the biosensing interface. Second, because tile rigid scaffold that allows the incorporation of dif- of their internal hybridized nature, these dynamic ferent soft functional nucleic acids into one or two probes are inherently resistant to inter-probe in- edges of the 3D structure, thereby forming dynamic teractions at the interface. Third, thermodynamic structures in response to different target molecules studies with foldable probes (single- or triple-stem [109,110]. DNA-conjugated protein probes should structure) have demonstrated their capability to sen- be explored, which can combine the advantages of sitively discriminate single-base mismatched DNA. each other (e.g. high stability of DNA and rapid However, it should be noted that foldable probes are binding rate of antigen antibody), and meanwhile not sufficiently rigid to survive on highly crowded offset shortcomings in part, like the instability of surfaces; hence, appropriate modulation of the sur- proteins in practical use and regeneration . face density is still critically important for high- Of note, the synergistic complex structure com- performance sensors. In addition, MCH is still re- posed of DNA nanostructure and antibody (TDN- quired to help stem-loop structures to stand upright antibody) may provide additional advantages: (i) at the surface, which makes the surface inevitably a the framework of TDN serves as a rigid spacer that heterogeneous one. endows the antibody with an orderly upright ori- entation and solution-phase setting; (ii) the com- parative size between TDN and antibody (∼5nm) enables the protein probes to be immobilized with PERSPECTIVES a controlled lateral distance and favorable accessi- bility; (iii) through simple thermal denaturation of In this review, we summarize the biomolecular bridged DNA, the antibody layer could be regener- nanostructures-mediated interfacial engineering, in which proteins and nucleic acids are capable of en- ated, guaranteeing a renewable sensing interface. gineering the biosensing interface in static or dy- The purity of the assembled DNA nanostruc- namic manners to improve sensitivity and speci- tures or DNA-protein conjugates is another con- ficity. In particular, the precise gene manipulation cern, which is limited by the separation techniques, Downloaded from https://academic.oup.com/nsr/article/5/5/740/4850656 by DeepDyve user on 20 July 2022 752 Natl Sci Rev, 2018, Vol. 5, No. 5 REVIEW such as electrophoresis and high-performance liq- 7. Sage AT, Besant JD and Lam B et al. Ultrasensitive electro- uid chromatography. Hence, the approaches with chemical biomolecular detection using nanostructured micro- higher separation efficiency, for example, single- electrodes. Acc Chem Res 2014; 47: 2417–25. step magnetic isolation, are required to be devel- 8. Vasilyeva E, Lam B and Fang Z et al. Direct genetic analysis of oped in future. Moreover, advanced synthetic biol- ten cancer cells: tuning sensor structure and molecular probe ogy , smart bioresponsive materials  and design for efficient mRNA capture. Angew Chem Int Ed 2011; genome-editing technology (e.g. CRISPR-Cas9) 50: 4137–41.  might be exploited to design and modify 9. 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National Science Review – Oxford University Press
Published: Sep 1, 2018
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