Differentiating between fluorescence-quenching metal ions with polyfluorophore sensors built on a DNA backbone

A common problem in detecting metal ions with fluorescentchemosensors is the emission-suppressing effects of fluorescence-quenching metal ions. This quenching tendency makes it difficult to design sensors with turn-on signal, and differentiate between several metal ions that may yield a strong quenching response. To address these challenges, we investigate a new sensor design strategy, incorporating fluorophores and metal ligands as DNA base replacements in DNA-like oligomers, for generating a broader range of responses for quenching metal ions. The modular molecular design enabled rapid synthesis and discovery of sensors from libraries on PEG-polystyrene beads. Using this approach, water-soluble sensors 1-5 were identified as strong responders to a set of eight typically quenching metal ions (Co(2+), Ni(2+), Cu(2+), Hg(2+), Pb(2+), Ag(+), Cr(3+), and Fe(3+)). They were synthesized and characterized for sensing responses in solution. Cross-screening with the full set of metal ions showed that they have a wide variety of responses, including emission enhancements and red- and blue-shifts. The diversity of sensor responses allows as few as two sensors (1 and 2) to be used together to successfully differentiate these eight metals. As a test, a set of unknown metal ion solutions in blind studies were also successfully identified based on the response pattern of the sensors. The modular nature of the sensor design strategy suggests a broadly applicable approach to finding sensors for differentiating many different cations by pattern-based recognition, simply by varying the sequence and composition of ligands and fluorophores on a DNA synthesizer.     

Genetically Encoded Ratiometric RNA‐Based Sensors for Quantitative Imaging of Small Molecules in Living Cells

Precisely determining the intracellular concentrations of metabolites and signaling molecules is critical in studying cell biology. Fluorogenic RNA‐based sensors have emerged to detect various targets in living cells. However, it is still challenging to apply these genetically encoded sensors to quantify the cellular concentrations and distributions of targets. Herein, using a pair of orthogonal fluorogenic RNA aptamers, DNB and Broccoli, we engineered a modular sensor system to apply the DNB‐to‐Broccoli fluorescence ratio to quantify the cell‐to‐cell variations of target concentrations. These ratiometric sensors can be broadly applied for live‐cell imaging and quantification of metabolites, signaling molecules, and other synthetic compounds.     

Chiral donor photoinduced-electron-transfer (d-PET) boronic acid chemosensors for the selective recognition of tartaric acids, disaccharides, and ginsenosides

A modular approach was proposed for the preparation of chiral fluorescent molecular sensors, in which the fluorophore, scaffold, and chirogenic center can be connected by ethynyl groups, and these modules can easily be changed to other structures to optimize the molecular sensing performance of the sensors. This modular strategy to assembly chiral sensors alleviated the previous restrictions of chiral boronic acid sensors, for which the chirogenic center, fluorophore, and scaffold were integrated, thus it was difficult to optimize the molecular structures by chemical modifications. We demonstrated the potential of our new strategy by the preparation of a sensor with a larger scaffold. The photoinduced electron‐transfer (PET) effect is efficient even with a large distance between the N atom and the fluorophore core. Furthermore, the rarely reported donor‐PET (d‐PET) effect, which was previously limited to carbazole, was extended to phenothiazine fluorophore. The contrast ratio, that is, PET efficiency of the new d‐PET sensor, is increased to 8.0, compared to 2.0 with the previous carbazole d‐PET sensors. Furthermore, the ethynylated phenothiazine shows longer excitation wavelength (centered at 380 nm) and emission wavelength (492 nm), a large Stokes shift (142 nm), and high fluorescence quantum yield in aqueous solution (Φ=0.48 in MeOH/water, 3:1 v/v). Enantioselective recognition of tartaric acid was achieved with the new d‐PET boronic acid sensors. The enantioselectivity is up to 10 (ratio of the binding constants toward D ‐ and L ‐tartaric acid, k_D/k_L). A consecutive fluorescence enhancement/decrease was observed, thus we propose a transition of the binding stoichiometry from 1:1 to 1:2 as the analyte concentration increases, which is supported by mass spectra analysis. The boronic acid sensors were used for selective and sensitive recognition of disaccharides and glycosylated steroids (ginsenosides).     

Label‐Free Logic Modules and Two‐Layer Cascade Based on Stem‐Loop Probes Containing a G‐Quadruplex Domain

A simple, versatile, and label‐free DNA computing strategy was designed by using toehold‐mediated strand displacement and stem‐loop probes. A full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two‐layer logic cascade were constructed. The probes contain a G‐quadruplex domain, which was blocked or unfolded through inputs initiating strand displacement and the obviously distinguishable light‐up fluorescent signal of G‐quadruplex/NMM complex was used as the output readout. The inputs are the disease‐specific nucleotide sequences with potential for clinic diagnosis. The developed versatile computing system based on our label‐free and modular strategy might be adapted in multi‐target diagnosis through DNA hybridization and aptamer‐target interaction.     

Harnessing Effective Molarity to Design an Electrochemical DNA‐based Platform for Clinically Relevant Antibody Detection

Easy‐to‐use platforms for rapid antibody detection are likely to improve molecular diagnostics and immunotherapy monitoring. However, current technologies require multi‐step, time‐consuming procedures that limit their applicability in these fields. Herein, we demonstrate effective molarity‐driven electrochemical DNA‐based detection of target antibodies. We show a highly selective, signal‐on DNA‐based sensor that takes advantage of antibody‐binding‐induced increase of local concentration to detect clinically relevant antibodies in blood serum. The sensing platform is modular, rapid, and versatile and allows the detection of both IgG and IgE antibodies. We also demonstrate the possible use of this strategy for the monitoring of therapeutic monoclonal antibodies in body fluids. Our approach highlights the potential of harnessing effective molarity for the design of electrochemical sensing strategies.     

Analyte‐Triggered DNA‐Probe Release from a Triplex Molecular Beacon for Nanopore Sensing

A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a triplex‐forming molecular beacon and a stem‐forming DNA component that is modified with a host–guest complex. Upon target DNA hybridizing with the molecular beacon loop or multivalent proteins binding to the recognition elements on the stem, the DNA probe is released and produces highly characteristic current signals when translocated through α‐hemolysin. The frequency of current signatures can be used to quantify the concentrations of the target molecules. This sensing approach provides a simple, quick, and modular tool for the detection of specific macromolecules with high sensitivity and excellent selectivity. It may find useful applications in point‐of‐care diagnostics with a portable nanopore kit in the future.     

Cytoprotective Silica Coating of Individual Mammalian Cells through Bioinspired Silicification

The cytoprotective coating of physicochemically labile mammalian cells with a durable material has potential applications in cell‐based sensors, cell therapy, and regenerative medicine, as well as providing a platform for fundamental single‐cell studies in cell biology. In this work, HeLa cells in suspension were individually coated with silica in a cytocompatible fashion through bioinspired silicification. The silica coating greatly enhanced the resistance of the HeLa cells to enzymatic attack by trypsin and the toxic compound poly(allylamine hydrochloride), while suppressing cell division in a controlled fashion. This bioinspired cytocompatible strategy for single‐cell coating was also applied to NIH 3T3 fibroblasts and Jurkat cells.     

Development of a New Label‐free,Indicator‐free Strategy toward Ultrasensitive Electrochemical DNA Biosensing Based on Fe3O4 Nanoparticles/Reduced Graphene Oxide Composite

Electrochemistry offers sensitivity, selectivity and low cost for fabrication of sensors capable of detection of selected DNA targets or mutated genes associated with human disease. In this work, we have developed a novel label‐free, indicator‐free strategy of electrochemical DNA sensor based on Fe₃O₄ nanoparticles/reduced graphene oxide (Fe₃O₄/r‐GO) nanocomposite modified electrode. By using Fe₃O₄/r‐GO nanocomposite as a substrate to immobilize probe DNA and subsequent hybridization with target sequence to form dsDNA, a great signal amplification was achieved through measuring changes in DPV peak current of underlying Fe(II)/Fe(III) redox system. With the remarkable attomolar sensitivity and high specificity and at the same time, great simplicity, the proposed strategy may find great applications in different DNA assay fields.     

An effective DNA-based electrochemical switch for reagentless detection of living cells

A novel switch-cytosensing strategy has been designed, which is based on the changes of electron transfer efficiency between ferrocene at the end of DNA and the electrode surface before and after cell transfection. This strategy provided a general and convenient method to fabricate efficient electrochemical cell sensors.     

Unit cloning and amplification as novel and universal strategies for complex vector construction and small DNA fragment preparation

With a novel and universal strategy for the cloning of multiple DNA fragments, a complex synthetic vector (pVEC100), harboring the target DNA fragments in conventional 100‐bp DNA ladder, was constructed for efficient and large‐scale production of 100‐bp DNA marker through bacteria fermentation, plasmid extraction and restrictive digestion. Since the restrictive digestion of complex vectors yields insufficient small DNA fragments, an innovative PCR model was developed as an alternative. The PCR model comprised a specially designed template vector and a unit amplification model for producing groups of small DNA fragments. The unit amplification model improved the efficiency of the PCR protocol and made it more economical and easier for small DNA fragment amplification. The approach presented in this paper – a unit cloning model for constructing complex synthetic vectors combined with the modular design of unit amplification by PCR – is a powerful method for preparing small DNA fragments of DNA molecular weight standards.     

Click To Bind: Metal Sensors

The copper‐catalyzed azide–alkyne “click” cycloaddition reaction is an efficient coupling reaction that results in the formation of a triazole ring. The wide range of applicable substrates for this reaction allows the construction of a variety of conjugated systems. The additional function of triazoles as metal‐ion ligands has led to the click reaction being used for the construction of optical sensors for metal ions. The triazoles are integral binding elements, which are formed in an efficient modular synthesis. Herein, we review recent examples of triazoles as a metal‐binding element in conjugated metal‐ion sensors.     

An Autonomous Bio‐barcode DNA Machine for Exponential DNA Amplification and Its Application to the Electrochemical Determination of Adenosine Triphosphate

A novel autonomous bio‐barcode DNA machine that is driven by template‐dependent DNA replication is developed to exponentially amplify special DNA sequences. Combined with a DNA aptamer recognition element, the DNA machine can be further applied in the aptamer‐based, amplified analysis of small molecules. As a model analyte, adenosine triphosphate (ATP) is determined by using the DNA machine system in combination with a DNA aptamer recognition strategy and differential pulse anodic stripping voltammetry (DPASV). Under the optimum conditions, detection limits as low as 2.8×10^?17 M (3σ) for target DNA and 4.7×10^?9 M (3σ) for ATP are achieved. The satisfactory determination of ATP in K562 leukemia cell and Ramos Burkitt’s lymphoma cell reveal that this protocol possesses good selectivity and practicality. As a promising biomolecular device, this DNA machine may have an even broader application in the rapidly developing field of nanobiotechnology.     

Practical Chemical Synthesis of Atypical Ubiquitin Chains by Using an Isopeptide‐Linked Ub Isomer

Chemical ubiquitination is an effective approach for accessing structurally defined, atypical ubiquitin (Ub) chains that are difficult to prepare by other techniques. Herein, we describe a strategy that uses a readily accessible premade isopeptide‐linked 76‐mer (isoUb), which has an N‐terminal Cys and a C‐terminal hydrazide, as the key building block to assemble atypical Ub chains in a modular fashion. This method avoids the use of auxiliary‐modified Lys and instead employs the canonical and therefore more robust Cys‐based native chemical ligation technique. The efficiency and capacity of this isoUb‐based strategy is exemplified by the cost‐effective synthesis of several linkage‐ and length‐defined atypical Ub chains, including K27‐linked tetra‐Ub and K11/K48‐branched tri‐, tetra‐, penta‐, and hexa‐Ubs.     

Modification of polysaccharides via thiol‐ene chemistry: A versatile route to functional biomaterials

We present herein a mild and rapid method for the modular functionalization of polysaccharides. Several ene‐functional charged and neutral polysaccharides, that is, hyaluronic acid and dextran, were prepared by esterification of the hydroxyl groups with pentenoic anhydride. The modified polysaccharides were then reacted with six model mercaptans under UV light, leading to linear polymers modified with hydrophobic groups, peptides, or oligosaccharides as well as chemical hydrogels. The thiol‐ene coupling reactions were found to proceed with high efficiency in short reaction times and with nearly no degradation of the polysaccharide backbone. Moreover, they were carried out in aqueous media, without the use of any metal catalysts, enhancing the attractive nature of this process. Notably, we investigated whether it is feasible to prepare cell‐responsive hydrogels by sequential bioconjugation and cross‐linking of the polysaccharide backbone with a bioactive peptide and poly(ethylene glycol)‐dithiol, respectively. All together, these results highlight the potential of this coupling strategy for the modular functionalization of polysaccharides under click chemistry‐like conditions. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Sequence‐Independent DNA Nanogel as a Potential Drug Carrier

DNA nanostructures largely rely on pairing DNA bases; thus, sequence designing is required. Here, this study demonstrates a sequence‐independent strategy to fabricate DNA nanogel (NG) inspired by cisplatin, a chemotherapeutic drug that acts as a DNA crosslinker. A simple heating and cooling of the genomic DNA extracts and cisplatin produces DNA NG with a size controlled by the heating time. Furthermore, the drug‐loaded NG is formulated by spontaneously mixing DNA segments, cisplatin, and doxorubicin. The in vitro cell studies demonstrate that the doxorubicin‐loaded NG alters the drug distribution in cells while its cytotoxic potential is well‐maintained. This chemotherapeutic‐inspired method provides a facile one‐pot and cost‐effective strategy to fabricate size‐controllable DNA NG that potentially acts as drug carrier.     

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.     

Versatile Logic Devices Based on Programmable DNA‐Regulated Silver‐Nanocluster Signal Transducers

A DNA‐encoding strategy is reported for the programmable regulation of the fluorescence properties of silver nanoclusters (AgNCs). By taking advantage of the DNA‐encoding strategy, aqueous AgNCs were used as signal transducers to convert DNA inputs into fluorescence outputs for the construction of various DNA‐based logic gates (AND, OR, INHIBIT, XOR, NOR, XNOR, NAND, and a sequential logic gate). Moreover, a biomolecular keypad that was capable of constructing crossword puzzles was also fabricated. These AgNC‐based logic systems showed several advantages, including a simple transducer‐introduction strategy, universal design, and biocompatible operation. In addition, this proof of concept opens the door to a new generation of signal transducer materials and provides a general route to versatile biomolecular logic devices for practical applications.     

Graphene Paper Decorated with a 2D Array of Dendritic Platinum Nanoparticles for Ultrasensitive Electrochemical Detection of Dopamine Secreted by Live Cells

To circumvent the bottlenecks of non‐flexibility, low sensitivity, and narrow workable detection range of conventional biosensors for biological molecule detection (e.g., dopamine (DA) secreted by living cells), a new hybrid flexible electrochemical biosensor has been created by decorating closely packed dendritic Pt nanoparticles (NPs) on freestanding graphene paper. This innovative structural integration of ultrathin graphene paper and uniform 2D arrays of dendritic NPs by tailored wet chemical synthesis has been achieved by a modular strategy through a facile and delicately controlled oil–water interfacial assembly method, whereby the uniform distribution of catalytic dendritic NPs on the graphene paper is maximized. In this way, the performance is improved by several orders of magnitude. The developed hybrid electrode shows a high sensitivity of 2 μA cm^?2 μm ^?1, up to about 33 times higher than those of conventional sensors, a low detection limit of 5 nm, and a wide linear range of 87 nm to 100 μm . These combined features enable the ultrasensitive detection of DA released from pheochromocytoma (PC 12) cells. The unique features of this flexible sensor can be attributed to the well‐tailored uniform 2D array of dendritic Pt NPs and the modular electrode assembly at the oil–water interface. Its excellent performance holds much promise for the future development of optimized flexible electrochemical sensors for a diverse range of electroactive molecules to better serve society.     

FET‐Based Biosensors for The Direct Detection of Organophosphate Neurotoxins

Recent world‐wide terrorist events associated with the threat of hazardous chemical agent proliferation, and outbreaks of chemical contamination in the food supply has demonstrated an urgent need for sensors that can directly detect the presence of dangerous chemical toxins. Such sensors must enable real‐time detection and accurate identification of different classes of pesticides (e.g., carbamates and organophosphates) but must especially discriminate between widely used organophosphate (OP) pesticides and G‐ and V‐type organophosphate chemical warfare nerve agents. Present field analytic sensors are bulky with limited specificity, require specially‐trained personnel, and, in some cases, depend upon lengthy analysis time and specialized facilities. Most bioanalytical based systems are biomimetic. These sensors utilize sensitive enzyme recognition elements that are the in‐vivo target of the neurotoxic agents which the sensor is attempting to detect. The strategy is well founded; if you want to detect cholinesterase toxins use cholinesterase receptors. However, this approach has multiple limitations. Cholinesterase receptors are sensitive to a wide range of non‐related compounds and require lengthy incubation time. Cholinesterase sensors are inherently inhibition mode and therefore require baseline testing followed by sample exposure, retest and comparison to baseline. Finally, due to the irreversible nature of enzyme‐ligand interactions, inhibition‐mode sensors cannot be reused without regeneration of enzyme activity, which in many cases is inefficient and time‐consuming. In 1996, we pioneered a new “kinetic” approach for the direct detection of OP neurotoxins based on agent hydrolysis by the enzyme organophosphate hydrolase (OPH; EC 3.1.8.2; phosphotriesterase) and further identified a novel multi‐enzyme strategy for discrimination between different classes of neurotoxins. The major advantage of this sensor strategy is it allows direct and continuous measurement of OP agents using a reversible biorecognition element. We also investigated incorporation of enzymes with variations in substrate specificity (e.g., native OPH, site‐directed mutants of OPH, and OPAA (EC 3.1.8.1), based upon preferential hydrolysis of P? O, P? F and P? S bonds to enable discrimination among chemically diverse OP compounds. Organophosphate hydrolase enzymes were integrated with several different transduction platforms including conventional pH electrodes, fluoride ion‐sensitive electrodes, and pH‐responsive fluorescent dyes. Detection limit for most systems was in the low ppm concentration range. This article reviews our integration of organophosphate hydrolase enzymes with pH sensitive field effect transistors (FETs) for OP detection.     

Label‐free Electrochemiluminescent Enantioselective Sensor for Distinguishing between Chiral Metallosupramolecular Complexes

Chiral molecular recognition of DNA is important for rational drug design and for developing structural probes of DNA conformation. Developing a convenient and inexpensive assay for sensitive and selective identification of DNA‐specific binding compounds with rapid, easy manipulation is in ever‐increasing demand. Here, we present a “turn‐on” and label‐free electrochemiluminescent (ECL) biosensor for distinguishing chiral metallosupramolecular complexes based on DNA three‐way junction formation selectively induced by the analyte. The fabricated ECL sensor shows excellent performance in the chiral discrimination of two enantiomers with an enantioselective recognition ratio of up to 4.4. More importantly, as a “turn‐on” detection system, the ECL chiral sensor does not suffer from false positives and limited signal range of “signal‐off” systems. Therefore, this concept may provide a new insight into the design of efficient sensors for distinguishing chiral molecules and for investigating the interactions between DNA and small molecules.