A Molecular Tuning Fork in Single‐Molecule Mechanochemical Sensing

The separate arrangement of target recognition and signal transduction in conventional biosensors often compromises the real‐time response and can introduce additional noise. To address these issues, we combined analyte recognition and signal reporting by mechanochemical coupling in a single‐molecule DNA template. We incorporated a DNA hairpin as a mechanophore in the template, which, under a specific force, undergoes stochastic transitions between folded and unfolded hairpin structures (mechanoescence). Reminiscent of a tuning fork that vibrates at a fixed frequency, the device was classified as a molecular tuning fork (MTF). By monitoring the lifetime of the folded and unfolded hairpins with equal populations, we were able to differentiate between the mono‐ and bivalent binding modes during individual antibody‐antigen binding events. We anticipate these mechanospectroscopic concepts and methods will be instrumental for the development of novel bioanalyses.     

Single‐Molecule Mechanochemical Sensing Using DNA Origami Nanostructures

While single‐molecule sensing offers the ultimate detection limit, its throughput is often restricted as sensing events are carried out one at a time in most cases. 2D and 3D DNA origami nanostructures are used as expanded single‐molecule platforms in a new mechanochemical sensing strategy. As a proof of concept, six sensing probes are incorporated in a 7‐tile DNA origami nanoassembly, wherein binding of a target molecule to any of these probes leads to mechanochemical rearrangement of the origami nanostructure, which is monitored in real time by optical tweezers. Using these platforms, 10 pM platelet‐derived growth factor (PDGF) are detected within 10 minutes, while demonstrating multiplex sensing of the PDGF and a target DNA in the same solution. By tapping into the rapid development of versatile DNA origami nanostructures, this mechanochemical platform is anticipated to offer a long sought solution for single‐molecule sensing with improved throughput.     

DNA‐Based Nanopore Sensing

Nanopore sensing is an attractive, label‐free approach that can measure single molecules. Although initially proposed for rapid and low‐cost DNA sequencing, nanopore sensors have been successfully employed in the detection of a wide variety of substrates. Early successes were mostly achieved based on two main strategies by 1) creating sensing elements inside the nanopore through protein mutation and chemical modification or 2) using molecular adapters to enhance analyte recognition. Over the past five years, DNA molecules started to be used as probes for sensing rather than substrates for sequencing. In this Minireview, we highlight the recent research efforts of nanopore sensing based on DNA‐mediated characteristic current events. As nanopore sensing is becoming increasingly important in biochemical and biophysical studies, DNA‐based sensing may find wider applications in investigating DNA‐involving biological processes.     

Sensing through signal amplification

The naked eye detection of single molecules in a complex mixture is the ultimate detection limit. Since a single molecule is unable to generate a strong enough signal, sensing methodologies able to reach that limit by necessity need to rely on signal amplification. This tutorial review describes various molecular approaches towards signal amplification in which a single analyte molecule affects the properties of a multitude of reporter molecules. Sensing by advanced instrumentation or changes in the physical properties of materials are excluded. The review is divided into four parts (catalysts, macromolecules, metal surfaces and supramolecular aggregates) depending on the species responsible for generating reporter molecules. Although on first sight apparently very diverse in nature, the majority of approaches rely on two key concepts: catalysis and multivalency. The ability of a catalyst to convert a multitude of substrate molecules into product (defined by the turn over number) makes a catalyst an intrinsic signal amplifier in case the chemical conversion of the substrate is accompanied by a measurable change in physical properties. For sensing purposes, catalytic activity must depend on the interaction between the analyte and the catalyst. Sensing using multivalent structures such as polymers and functionalized nanoparticles relies on the ability of a single analyte molecule to affect the properties of a multitude of reporter molecules collected in the multivalent structure. Chemical sensing systems will be discussed with detection limits that indeed go down to a few molecules and can rival the best biological assays. It will be shown that the most sensitive methods rely on a cascade of amplification mechanisms.     

Adaptive Supramolecular Networks: Emergent Sensing from Complex Systems

Molecular differentiation by supramolecular sensors is typically achieved through sensor arrays, relying on the pattern recognition responses of large panels of isolated sensing elements. Here we report a new one-pot systems chemistry approach to differential sensing in biological solutions. We constructed an adaptive network of three cross-assembling sensor elements with diverse analyte-binding and photophysical properties. This robust sensing approach exploits complex interconnected sensor-sensor and sensor-analyte equilibria, producing emergent supramolecular and photophysical responses unique to each analyte. We characterize the basic mechanisms by which an adaptive network responds to analytes. The inherently data-rich responses of an adaptive network discriminate among very closely related proteins and protein mixtures without relying on designed protein recognition elements. We show that a single adaptive sensing solution provides better analyte discrimination using fewer response observations than a sensor array built from the same components. We also show the network's ability to adapt and respond to changing biological solutions over time.     

Noncovalent Chirality Sensing Ensembles for the Detection and Reaction Monitoring of Amino Acids,Peptides, Proteins,and Aromatic Drugs

Ternary complexes between the macrocyclic host cucurbit[8]uril, dicationic dyes, and chiral aromatic analytes afford strong induced circular dichroism (ICD) signals in the near‐UV and visible regions. This allows for chirality sensing and peptide‐sequence recognition in water at low micromolar analyte concentrations. The reversible and noncovalent mode of binding ensures an immediate response to concentration changes, which allows the real‐time monitoring of chemical reactions. The introduced supramolecular method is likely to find applications in bioanalytical chemistry, especially enzyme assays, for drug‐related analytical applications, and for continuous monitoring of enantioselective reactions, particularly asymmetric catalysis.     

Detection of Explosive Vapors: The Roles of Exciton and Molecular Diffusion in Real‐Time Sensing

Time‐resolved quartz crystal microbalance with in situ fluorescence measurements are used to monitor the sorption of the nitroaromatic (explosive) vapor, 2,4‐dinitrotoluene (DNT) into a porous pentiptycene‐containing poly(phenyleneethynylene) sensing film. Correlation of the nitroaromatic mass uptake with fluorescence quenching shows that the analyte diffusion follows the Case‐II transport model, a film‐swelling‐limited process, in which a sharp diffusional front propagates at a constant velocity through the film. At a low vapor pressure of DNT of ≈16 ppb, the analyte concentration in the front is sufficiently high to give an average fluorophore–analyte separation of ≈1.5 nm. Hence, a long exciton diffusion length is not required for real‐time sensing in the solid state. Rather the diffusion behavior of the analyte and the strength of the binding interaction between the analyte and the polymer play first‐order roles in the fluorescence quenching process.     

Deposition Strategies for Osmium/Enzyme Films on Gold Electrode Based Sensing Arrays

Multi‐analyte real‐time interrogation of cellular activity allows for the potential discovery of novel insights into disease. In this report, the addition of electrochemical biosensors to a previously developed platform utilizing Au microfabricated electrodes was explored. Glucose oxidase was immobilized at the electrode surface with an osmium redox polymer, using hand‐casting and electrodeposition techniques, allowing for the first comparison of deposition techniques at a Au microfabricated array. This preliminary work is the first step toward the goal of creating a multi‐analyte array for cellular analysis.     

Application and analysis of structure-switching aptamers for small molecule quantification

Modern tools for the analysis of cellular function aim for the quantitative measurement of all members of a given class of biological molecules. Of the analyte classes, nucleic acid measurements are typically the most tractable, both on an individual analyte basis and in parallel. Thus, tools are being sought to enable measurement of other cellular molecules using nucleic acid biosensors. Of the variety of potential nucleic acid biosensor strategies, structure-switching aptamers (SSAs) present a unique opportunity to couple sensing and readout of the target molecule. However, little has been characterized about the parameters that determine the fidelity of the signal from SSA biosensors. In this study, a small molecule biosensor based on a SSA was engineered to detect the model small molecule, theophylline, in solution. Quantitative theophylline detection over nearly three orders-of-magnitude was achieved by scintillation counting and quantitative PCR. Further analysis showed that the biosensor fidelity is primarily controlled by the relative stability of the two conformations of the SSA.     

Toward Sol-Gel-Processed Chemical Sensing Platforms: Effects of Dopant Addition Time on Sensor Performance

The development of new chemical and biochemical sensing schemes has been a topic of growing interest. Simplicity of preparation and mild processing conditions have made sol-gel-derived composites attractive for many chemical sensing schemes. A portion of our research centers on using sol-gel-processed materials for the development of selective sensors. Over the years we have aimed to characterize the analytical performance of these types of sol-gel-based sensing platforms. In the course of this work we recently discovered that the time (prior to casting) when the sensing chemistry is actually doped into the sol-gel processing solution plays a critical role in a given sensor's analytical performance. In this paper we report on the effects of doping time on the behavior of a model organic dopant (pyrene) sequestered within sol-gel-derived microfiber tips and films. We use O₂ as the analyte and determine the sensor sensitivity and temporal response as a function of doping time. We also quantify the local dipolarity of the immediate environment surrounding the average pyrene molecule as a function of doping time.     

A ligation-triggered DNAzyme cascade for amplified fluorescence detection of biological small molecules with zero-background signal

Many types of fluorescent sensing systems have been reported for biological small molecules. Particularly, several methods have been developed for the recognition of ATP or NAD(+), but they only show moderate sensitivity, and they cannot discriminate either ATP or NAD(+) from their respective analogues. We have addressed these limitations and report here a dual strategy which combines split DNAzyme-based background reduction with catalytic and molecular beacon (CAMB)-based amplified detection to develop a ligation-triggered DNAzyme cascade, resulting in ultrahigh sensitivity. First, the 8-17 DNAzyme is split into two separate oligonucleotide fragments as the building blocks for the DNA ligation reaction, thereby providing a zero-background signal to improve overall sensitivity. Next, a CAMB strategy is further employed for amplified signal detection achieved through cycling and regenerating the DNAzyme to realize the true enzymatic multiple turnover (one enzyme catalyzes the cleavage of several substrates) of catalytic beacons. This combination of zero-background signal and signal amplification significantly improves the sensitivity of the sensing systems, resulting in detection limits of 100 and 50 pM for ATP and NAD(+), respectively, much lower than those of previously reported biosensors. Moreover, by taking advantage of the highly specific biomolecule-dependence of the DNA ligation reaction, the developed DNAzyme cascades show significantly high selectivity toward the target cofactor (ATP or NAD(+)), and the target biological small molecule can be distinguished from its analogues. Therefore, as a new and universal platform for the design of DNA ligation reaction-based sensing systems, this novel ligation-triggered DNAzyme cascade method may find a broad spectrum of applications in both environmental and biomedical fields.     

Molecularly Imprinted Photonic Polymers as Sensing Elements for the Creation of Cross‐Reactive Sensor Arrays

By combining molecular imprinting and colloidal crystal templating, molecularly imprinted inverse‐opal photonic polymers (MIPPs) acting as sensing elements have been exploited to create sensor arrays for the first time. With this new strategy, abundant sensing elements with differential sensing abilities were easily accessible. Because of the unique hierarchical porous structure integrated in each sensing element, high sensitivity and selectivity, fast response and self‐reporting (label‐free) detection could be simultaneously achieved. All these fascinating features indicate that MIPPs are ideal sensing elements for creating sensor arrays. By integrating the individual sensing elements on a substrate, the formed array chip delivers better portability and high‐throughput capability. As a demonstration, six kinds of contaminants were selected as analytes. The detection and discrimination of these analytes and even their mixtures in a wide range of concentrations, particularly trace amounts of analyte against a high background of other components, could be achieved, indicating the powerful capability of MIPPs‐based sensor array for sensing. These results suggest that the described strategy opens a new route for sensor array creation and should find important applications in a wide range of areas.     

Sacrificial BSA to block non‐specific adsorption on organosilane adlayers in ultra‐high frequency acoustic wave sensing

This follow‐up study describes the implementation of recently developed cross‐linking trichlorosilane surface chemistry with acoustic wave sensing technology for the real‐time and label‐free detection of biotin/avidin interactions. Biosensing platforms consist of unelectroded piezoelectric quartz resonator discs onto which functionalizable mixed organosilane adlayers are prepared using a new trichlorosilane cross‐linker in combination with a shorter monofunctional diluent molecule. Thiolated or aminated biotin probes can next be anchored to the mixed assembly in a single, preactivation‐free step through site‐specific coupling at pentafluorophenyl ester head moieties. Biosensing properties are assessed at ultra‐high frequency (>0.74 GHz) with the highly sensitive electromagnetic piezoelectric acoustic sensor using micromolar buffered solutions of avidin. This biosensor prototype – which generally displays good reproducibility – uses sacrificial bovine serum albumin to block non‐specific adsorption. This preliminary work in buffer constitutes an important step towards the development of real‐world biosensors able to perform with more demanding biological samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Nano‐Graphene Oxide Based Multichannel Sensor Arrays towards Sensing of Protein Mixtures

Optical array‐based sensors are attractive candidates for the detection of various bio‐analytes due to their convenient fabrication and measurements. For array‐based sensors, multichannel arrays are more advantageous and used frequently in many electronic sensors. But most reported optically array based sensors are constructed on a single channel array. This difficulty is mainly instigated from the overlap in optical responses. In this report we have used nano‐graphene oxide (nGO) and suitable fluorophores as sensor elements to construct a multichannel sensor array for the detection of protein analytes. By using the optimized multichannel array we are able to detect different proteins and mixtures of proteins with 100 % classification accuracy at sub‐nanomolar concentration. This modified method expedites the sensing analysis as well as minimizes the use of both analyte and sensor elements in array‐based protein sensing. We have also used this system for the single channel array‐based sensing to compare the sensitivity and the efficacy of these two systems for other applications. This work demonstrated an intrinsic trade‐off associated with these two methods which may be necessary to balance for array‐based analyte detections.     

Protein biosensors based on biofunctionalized conical gold nanotubes

There is increasing interest in the concept of using nanopores as the sensing elements in biosensors. The nanopore most often used is the alpha-hemolysin protein channel, and the sensor consists of a single channel embedded within a lipid bilayer membrane. An ionic current is passed through the channel, and analyte species are detected as transient blocks in this current associated with translocation of the analyte through the channel-stochastic sensing. While this is an extremely promising sensing paradigm, it would be advantageous to eliminate the very fragile lipid bilayer membrane and perhaps to replace the biological nanopore with an abiotic equivalent. We describe here a new family of protein biosensors that are based on conically shaped gold nanotubes embedded within a mechanical and chemically robust polymeric membrane. While these sensors also function by passing an ion current through the nanotube, the sensing paradigm is different from the previous devices in that a transient change in the current is not observed. Instead, the protein analyte binds to a biochemical molecular-recognition agent at the mouth of the conical nanotube, resulting in complete blockage of the ion current. Three different molecular-recognition agents, and correspondingly three different protein analytes, were investigated: (i) biotin/streptavidin, (ii) protein-G/immunoglobulin, and (iii) an antibody to the protein ricin with ricin as the analyte.     

Recent Advances in Luminescent Recognition and Chemosensing of Iodide in Water

This Minireview covers the latest developments of chemosensors based on transition‐metal receptors and organic fluorophores with specific binding sites for the luminescent detection and recognition of iodide in aqueous media and real samples. In all selected examples within the last decade (made‐post 2010), the iodide sensing and recognition is probed by monitoring real‐time changes of the fluorescence or phosphorescence properties of the chemosensors. This review highlights effective strategies to iodide sensing from a structural approach where the iodide recognition/sensing process, through supramolecular interactions as coordination bonds, hydrogen bonds, halogen bonds and electrostatic interactions, is transduced into an optical change easily measurable. The selective iodide sensing is an active field of research with global interest due to the importance of iodide in biological, medicinal, industrial, environmental and chemical processes.     

Polymers for Anion Recognition and Sensing

In biological systems, the selective and high‐affinity recognition of anionic species is accomplished by macromolecular hosts (anion‐binding proteins) that have been “optimized” through evolution. Surprisingly, it is only recently that chemists have systematically attempted to develop anion‐responsive synthetic macromolecules for potential applications in medicine, national security, or environmental monitoring. Recent results indicating that unique features of polymeric systems such as signal amplification, multivalency, and cooperative behavior may be exploited productively in the context of anion recognition and sensing are documented. The wide variety of interactions—including Lewis acid/base, ion‐pairing, and hydrogen bonding—that have been employed for this purpose is reflected in the structural diversity of anion‐responsive macromolecules identified to date.     

Ionophore‐Based Biphasic Chemical Sensing in Droplet Microfluidics

Droplet microfluidics is an enabling platform for high‐throughput screens, single‐cell studies, low‐volume chemical diagnostics, and microscale material syntheses. Analytical methods for real‐time and in situ detection of chemicals in the droplets will benefit these applications, but they remain limited. Reported herein is a novel heterogeneous chemical sensing strategy based on functionalization of the oil phase with rationally combined sensing reagents. Sub‐nanoliter oil segments containing pH‐sensitive fluorophores, ionophores, and ion‐exchangers enable highly selective and rapid fluorescence detection of physiologically important electrolytes (K⁺, Na⁺, and Cl^?) and polyions (protamine) in sub‐nanoliter aqueous droplets. Electrolyte analysis in whole blood is demonstrated without suffering from optical interference from the sample matrix. Moreover, an oil phase doped with an aza‐BODIPY dye allows indication of H₂O₂ in the aqueous droplets, exemplifying sensing of targets beyond ionic species.     

Bicomponent Microneedle Array Biosensor for Minimally‐Invasive Glutamate Monitoring

This article describes the design of a new and attractive minimally‐invasive bicomponent microneedle sensing device for the electrochemical monitoring of the excitatory neurotransmitter glutamate and glucose. The new device architecture relies on the close integration of solid and hollow microneedles into a single biosensor array device containing multiple microcavities. Such microcavities facilitate the electropolymeric entrapment of the recognition enzyme within each microrecess. The resulting microneedle biosensor array can be employed as a minimally‐invasive on‐body transdermal patch, obviating the extraction/sampling of the biological fluid, thereby simplifying device requirements. The new concept is demonstrated for the electropolymeric entrapment of glutamate oxidase and glucose oxidase within a poly(o‐phenylenediamine) (PPD) thin film. The PPD‐based enzyme entrapment methodology enables the effective rejection of coexisting electroactive interferents without compromising the sensitivity or response time of the device. The resulting microneedle‐based glutamate and glucose biosensors thus exhibit high selectivity, sensitivity, speed, and stability in both buffer and undiluted human serum. High‐fidelity glutamate measurements down to the 10 µM level are obtained in serum. The attractive recess design also serves to protect the enzyme layer upon insertion into the skin. This simple, yet robust microneedle design is well‐suited for diverse biosensing applications in which real‐time metabolite monitoring is a core requirement.     

Carbon‐Nanotube Based Electrochemical Biosensors: A Review

This review addresses recent advances in carbon‐nanotubes (CNT) based electrochemical biosensors. The unique chemical and physical properties of CNT have paved the way to new and improved sensing devices, in general, and electrochemical biosensors, in particular. CNT‐based electrochemical transducers offer substantial improvements in the performance of amperometric enzyme electrodes, immunosensors and nucleic‐acid sensing devices. The greatly enhanced electrochemical reactivity of hydrogen peroxide and NADH at CNT‐modified electrodes makes these nanomaterials extremely attractive for numerous oxidase‐ and dehydrogenase‐based amperometric biosensors. Aligned CNT “forests” can act as molecular wires to allow efficient electron transfer between the underlying electrode and the redox centers of enzymes. Bioaffinity devices utilizing enzyme tags can greatly benefit from the enhanced response of the biocatalytic‐reaction product at the CNT transducer and from CNT amplification platforms carrying multiple tags. Common designs of CNT‐based biosensors are discussed, along with practical examples of such devices. The successful realization of CNT‐based biosensors requires proper control of their chemical and physical properties, as well as their functionalization and surface immobilization.