A dual-blocker aided and dual-label-free electrochemical biosensor based on mbHCR/rGO nanocomplexes for ultrasensitive DNA detection.

Heterogeneous electrochemical DNA biosensors have attracted huge attention due to their enhanced signal sensitivity, compared to homogeneous biosensors. However, the high cost of probe labeling and the reduced recognition efficiency associated with current heterogeneous electrochemical biosensors confine their potential applications. In the present work, a dual-blocker assisted and dual-label-free heterogeneous electrochemical strategy based on multi-branched hybridization chain reaction (mbHCR) and reduced graphene oxide (rGO) was fabricated for ultrasensitive detection of DNA. The target DNA could trigger the mbHCR of two DNA hairpin probes, resulting in the generation of multi-branched long chain of DNA duplexes with bidirectional arms. One direction of the multi-branched arms in the mbHCR products were then bound to the label-free capture probe on the gold electrode through multivalent hybridization with enhanced recognition efficiency. The other direction of multi-branched arms in mbHCR product could adsorb rGO via π-π stacking interactions. Two DNA blockers were ingeniously designed to block the binding of excessive H1-pAT on electrode and to prevent the adsorption of rGO by residual unbound capture probes. As a result, with the electrochemical reporter methylene blue selectively intercalated into the long chain of DNA duplex and absorbed on rGO, a remarkable electrochemical signal rise was observed. Thus, a dual-blocker aided and dual-label-free electrochemical strategy for ultrasensitive DNA detection is readily realized with the merit of cost-effective. The as-developed dual-label-free electrochemical biosensor has great potential to be employed in nucleic acid related medical diagnostics.

An entropy-driven signal-off DNA circuit for label-free, visual detection of small molecules with enhanced accuracy.

An entropy-driven DNA circuit offers an efficient means of sensitive analyte detection with signal amplification. In this article, we rationally engineered an aptamer-based entropy-driven signal-off DNA circuit for colorimetric detection of small molecules. The proposed signal-off DNA circuit is activated by target small molecule binding to drive the collapse of G-quadruplex DNAzyme, accompanied by the colour change of the detection solution from dark blue to light blue. Entropy-driven recycling hybridization significantly magnified the input signal of the target small molecule. Such an assay enables naked-eye detection of adenosine triphosphate and oxytetracycline at concentrations as low as 0.5 µM and 1 µM respectively. Moreover, when compared with the signal-on DNA circuit, the entropy-driven signal-off DNA circuit for colorimetric detection has two advantages. Firstly, unlike in the signal-on DNA circuit, the unavoidable formation of waste complexes in the absence of a target in the signal-off DNA circuit has no influence on target detection performance as its background signal is only determined by the substrate complex. Secondly, the signal-on DNA circuit cannot distinguish false-positive signals generated by invasive catalysts (e.g., HRP, serum, Fe3O4), while the signal-off DNA circuit can distinguish those signals as undesired signals. Overall, the signal-off DNA circuit affords a novel strategy for sensitive and accurate detection of small molecules.

Enzymatic Cycle-Inspired Dynamic Biosensors Affording No False-Positive Identification.

There is an urgent need for reliable biosensors to detect nucleic acid of interest in clinical samples. We propose that the accuracy of the present nucleic acid-sensing method can be advanced by avoiding false-positive identifications derived from nonspecific interactions (e.g., nonspecific binding, probe degradation). The challenge is to exploit biosensors that can distinguish false-positive from true-positive samples in nucleic acid screening. In the present study, by learning from the enzymatic cycle in nature, we raise an allostery tool displaying invertible positive/negative cooperativity for reversible or cyclic activity control of the biosensing probe. We demonstrate that the silencing and regeneration of a positive (or negative) allosteric effector can be carried out through toehold displacement or an enzymatic reaction. We, thus, have developed several dynamic biosensors that can repeatedly measure a single nucleic acid sample. The ability to distinguish a false-positive from a true-positive signal is ascribed to the nonspecific interaction presenting equivalent signal variations, while the specific target binding exhibits diverse signal variations according to repeated measurements. Given its precise identification, such consequent dynamic biosensors offer exciting opportunities in physiological and pathological diagnosis.

Tunable Dual-Effector Allostery System for Nucleic Acid Analysis with Enhanced Sensitivity and an Extended Dynamic Range.

In the last few years, studies have demonstrated the existence of dual-effector allosteric cooperativity in nature and the mechanism underlying enhanced activation/inhibition performance. In this work, we design an artificial dual-effector allostery system for the construction of a dynamic biosensor that can achieve nucleic acid detection with superior sensitivity and across an extraordinary broad detection range. Our dual-effector allostery-regulated biosensor is based on the multibranched hybridization chain reaction (mHCR) involving three hairpins (H1, H2, and H3). In the presence of the target nucleic acid, the mHCR is initiated via cascading strand displacement events. The products of mHCR are then captured on the electrode surface based on the mechanism of the multivalent proximity ligation assay (mPLA) and the multivalent binding assay (mBA). The subsequent conjugation of streptavidin-modified horseradish peroxidase (SA-HRP) can lead to an increase in the electrochemical signal. Importantly, two distinct allosteric activation sites and two distinct allosteric inhibition sites in H1 are designed to fine-tune the nucleic acid detection sensitivity and the dynamic range. Using this new dual-effector allostery tool, we report the detection of nucleic acid at a dynamic range spanning 10-1012 aM, 11 orders of magnitude showing the broadest dynamic range reported to date with an allosteric regulation biosensor construct.

Highly Sensitive Determination of 2,4,6-Trichlorophenol by Using a Novel SiO2@MIPIL Fluorescence Sensor with a Double Recognition Functional Monomer.

A novel SiO2@ MIPIL fluorescence sensor for the highly sensitive detection of 2,4,6-trichlorophenol was prepared by using surface molecularly imprinting technology with SiO2 microspheres as carriers and 3,3'-(anthracene-9,10-diylbis(methylene))bis(1-vinyl-1H-imidazole-3-ium) chloride as a double recognition fluorescence functional monomer. The prepared molecularly imprinted polymer (SiO2@MIPIL) was characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, laser confocal microscopy, and nuclear magnetic resonance. Compared with the polymer obtained via bulk polymerization (MIPIL), the surface molecularly imprinted polymer (SiO2@MIPIL) has a better linear range (0.1-50 nM), lower detection limit (89 pM), and shorter detection time (approximately 1.5 min). The fluorescence sensor also shows good specificity, high sensitivity, good stability, and reusability. Satisfactory results were obtained when using this sensor in industrial wastewater and spiked environmental water.

A biosensor based on multifunctional allostery for dynamic analysis of circulating tumor DNA.

Here, a new strategy based on artificial multifunctional allostery (mFA) was explored to regulate the assembly of a DNA nanowire using circulating tumor DNA (ctDNA) as the initiator. Given its unique properties, the mFA-regulated versatile DNA nanowire was applied to engineer a single-step, amplified and dynamic biosensor for the quantitative analysis of ctDNA in serum samples with tunable sensitivity and selectivity.

One-pot ultrasonic synthesis of multifunctional Au nanoparticle-ferrocene-WS2 nanosheet composite for the construction of an electrochemical biosensing platform.

Structuring of noble metal nanoparticles on transition metal dichalcogenide nanosheets induces significantly enhanced electronic, optical, and catalytic functions. However, the synthesis of multifunctional hybrids is always time-consuming and involves multiple steps. Herein, a ternary Au nanoparticle-ferrocene-WS2 nanosheet (AFW) composite has been prepared by a facile one-step sonochemical approach. Stripped WS2 nanosheets were functionalized with ferrocene monocarboxylic acid (FMC) and gold nanoparticles (AuNPs) by making use of the strong coordinative interaction of carboxyl groups with tungsten atoms. The AuNPs decorating the WS2 nanosheet not only increase the water solubility of WS2 nanosheet and surface area of the modified electrode, but also act as electron transport bridges to aid the tunneling of electrons from the small redox molecule, FMC, through the space to the electrode on which they are mounted. Furthermore, the ternary AFW nanocomposite could effectively avoid the leaching of FMC from the nanocomposite matrix and provided a suitable environment for the immobilized biomolecules. Combining the immune magnetic beads technology and the AFW nanocomposite with aforementioned advantages, a high-performance electrochemical immunosensor was successfully developed using carbohydrate antigen 72-4 (CA72-4) as a model analyte. A linear relationship in the range of 2-50 U/L for the detection of CA72-4 was found with a low detection limit of 0.6 U/L. In addition, the biosensor showed excellent performance in selectivity, stability, and reproducibility. Thus, this work not only proposes a facile avenue for preparing a 2D WS2 nanocomposite with multifunctional properties but also opens up a new method to extend the application of WS2-based materials in biological sensing.

Fluorescent aptasensor based on D-AMA/F-CSC for the sensitive and specific recognition of myoglobin.

A novel fluorescent biosensor based on dabcyl [(E)-4-((4-(dimethylamino) phenyl) diazenyl)benzoic acid] -modified anti-Mb aptamer (D-AMA) and 6-FAM(6-carboxyfluorescein) -modified complementary short chain (F-CSC)for the specific and sensitive detection of Mb levels is presented in this study. In PBS buffer solution, D-AMA bound to F-CSC, and then dabcyl quenched the fluorescence of 6-FAM. After adding Mb into the system, D-AMA bound to Mb and separated from F-CSC. The fluorescence of 6-FAM was restored after it separated from dabcyl. The assay exhibited high specificity and sensitivity toward Mb, with a low limit of detection of 0.07 ng/mL (S/N = 3) and linear relationships of 0.1-5 ng/mL. It was further applied to detect Mb levels in spiked human blood sera samples.

Single-step multivalent capture assay for nucleic acid detection with dual-affinity regulation using mutation inhibition and allosteric activation.

The rational modulation of receptor affinity through distal-site mutation and allosteric control is valuable in biosensor designing to tune the useful dynamic range. Our ability to programmatically engineer dual-affinity regulation into diverse affinities of target binding and activities of hybridization chain reaction, however, remains limited. By programmable engineering of the switching equilibria of the recognition hairpin using distal-site mutation inhibition and allosteric activation, we obtained a set of receptors varying significantly in affinities of target binding and activities of the hybridization chain reaction. For the first time, we developed an electrocatalytic biosensor for nucleic acid detection with a tunable dynamic range based on a conformational switch triggered bidirectional hybridization chain reaction and blocker assisted multivalent binding. This designable biosensor thus enables single-step incubation, diverse affinities of target binding, diverse efficiencies of signal amplification and diverse single nucleotide discrimination for quantitative analyses of nucleic acids of various lengths in serum, which holds great potential as a compelling platform suitable for liquid biopsy.

Stem-loop clutch probes for sequence-specific dsDNA analysis with improved single-mismatch selectivity.

A stem-loop clutch probe (SLCP)-based strategy has been explored to guide sequence-specific double stranded DNA (dsDNA) analysis with enhanced single-base mismatch selectivity. This assay method can also modulate the dynamic range by employing natural processes relying on distal-site mutation inhibition and allosteric activation.

A Cross-Linker-Based Poly(Ionic Liquid) for Sensitive Electrochemical Detection of 4-Nonylphenol.

In this study, we report a cross-linker-based poly(ionic liquid) (PIL) for the sensitive detection of 4-nonylphenol (4-NP). PIL was poly(1,4-butanediyl-3,3'-bis-l-vinylimidazolium dibromide) (poly([V2C4(mim)2]Br2)). Poly([V2C4(mim)2]Br2) was prepared via one-step free-radical polymerization. The poly([V2C4(mim)2]Br2) was characterized by infrared spectroscopy, Raman spectroscopy, thermal gravimetric analyzer and scanning electron microscope. The poly([V2C4(mim)2]Br2) was then drop-cast onto a glassy carbon electrode (GCE) to obtain poly([V2C4(mim)2]Br2)/GCE. In comparison with a bare GCE, poly([V2C4(mim)2]Br2)/GCE exhibited higher peak current responses for [Fe(CN)6]3-/4-, lower charge transfer resistance, and larger effective surface area. While comparing the peak current responses, we found the peak current response for 4-NP using poly([V2C4(mim)2]Br2)/GCE to be 3.6 times higher than a traditional cross-linker ethylene glycol dimethacrylate (EGDMA) based poly(EGDMA) modified GCE. The peak current of poly([V2C4(mim)2]Br2) sensor was linear to 4-NP concentration from 0.05 to 5 µM. The detection limit of 4-NP was obtained as 0.01 µM (S/N = 3). The new PIL based electrochemical sensor also exhibited excellent selectivity, stability, and reusability. Furthermore, the poly([V2C4(mim)2]Br2)/GCE demonstrated good 4-NP detection in environmental water samples.

Fluorometric determination of cardiac myoglobin based on energy transfer from a pyrene-labeled aptamer to graphene oxide.

The authors describe a fluorometric assay for cardiac myoglobin (Mb), a marker for myocardial infarction. An Mb-binding aptamer was labeled with pyrene and adsorbed on the surface of graphene oxide (GO) via noncovalent and reversible binding forces. This causes the fluorescence of pyrene (best measured at excitation/emission wavelengths of 275/376 nm) to be quenched. However, fluorescence is restored on addition of pyrene due to the strong affinity between Mb and aptamer which causes its separation from GO. Fluorescence increases linearly in the 5.6-450 pM Mb concentration range, and the lower detection limit is 3.9 pM (S/N = 3). The assay was applied to the determination of cardiac Mb in spiked serum, and satisfactory results were obtained. Graphical abstract Schematic presentation of the detection of Mb (cardiac myoglobin) by using a fluorometric method based on pyrene-modified anti-Mb aptamer and GO (graphene oxide) through fluorescence quenching and subsequent recovery.

Colorimetric Method for Sensitive Detection of Microcystin-LR Using Surface Copper Nanoparticles of Polydopamine Nanosphere as Turn-On Probe.

A novel, facile sensor was further developed for microcystin-LR (MC-LR) determination by visible spectroscopy. Antibody-functionalized SiO2-coated magnetic nanoparticles (Fe3O4@SiO2) and aptamer-functionalized polydopamine nanospheres decorated with Cu nanoparticles (PDA/CuNPs) recognized specific sites in MC-LR and then the sandwich-type composites were separated magnetically. The Cu in the separated composites was converted to Cu2+ ions in solution and turn-on visible absorption was achieved after reaction with bis(cyclohexanone)oxaldihydrazone (BCO) (λmax = 600 nm). There was a quantitative relationship between the spectral intensity and MC-LR concentration. In addition, under the optimum conditions, the sensor turns out to be a linear relationship from 0.05 to 25 nM, with a limit of detection of 0.05 nM (0.05 µg/L) (S/N = 3) for MC-LR. The sensitivity was dependent on the low background absorption from the off-to-on spectrum and label amplification by the polydopamine (PDA) surface. The sensor had high selectivity, which shows the importance of dual-site recognition by the aptamer and antibody and the highly specific color formed by BCO with Cu2+. The bioassay was complete within 150 min, which enabled quick determination. The sensor was successfully used with real spiked samples. These results suggest it has potential applications in visible detection and could be used to detect other microcystin analogs.

Valency-Controlled Framework Nucleic Acid Signal Amplifiers.

Weak ligand-receptor recognition events are often amplified by recruiting multiple regulatory biomolecules to the action site in biological systems. However, signal amplification in in vitro biomimetic systems generally lack the spatiotemporal regulation in vivo. Herein we report a framework nucleic acid (FNA)-programmed strategy to develop valence-controlled signal amplifiers with high modularity for ultrasensitive biosensing. We demonstrated that the FNA-programmed signal amplifiers could recruit nucleic acids, proteins, and inorganic nanoparticles in a stoichiometric manner. The valence-controlled signal amplifier enhanced the quantification ability of electrochemical biosensors, and enabled ultrasensitive detection of tumor-relevant circulating free DNA (cfDNA) with sensitivity enhancement of 3-5 orders of magnitude and improved dynamic range.

Electrochemical detection of nucleic acids, proteins, small molecules and cells using a DNA-nanostructure-based universal biosensing platform.

The occurrence and prognosis of many complex diseases, such as cancers, is associated with the variation of various molecules, including DNA at the genetic level, RNA at the regulatory level, proteins at the functional level and small molecules at the metabolic level (defined collectively as multilevel molecules). Thus it is highly desirable to develop a single platform for detecting multilevel biomarkers for early-stage diagnosis. Here we report a protocol on DNA-nanostructure-based programmable engineering of the biomolecular recognition interface, which provides a universal electrochemical biosensing platform for the ultrasensitive detection of nucleic acids (DNA/RNA), proteins, small molecules and whole cells. The protocol starts with the synthesis of a series of differentially sized, self-assembled tetrahedral DNA nanostructures (TDNs) with site-specifically modified thiol groups that can be readily anchored on the surface of a gold electrode with high reproducibility. By exploiting the rigid structure, nanoscale addressability and versatile functionality of TDNs, one can tailor the type of biomolecular probes appended on individual TDNs for the detection of specific molecules of interest. Target binding occurring on the gold surface patterned with TDNs is quantitatively translated into electrochemical signals via a coupled enzyme-based catalytic process. This uses a sandwich assay strategy in which biotinylated reporter probes recognize TDN-bound target biomolecules, which then allow binding of horseradish-peroxidase-conjugated avidin (avidin-HRP). Hydrogen peroxide (H2O2) is then reduced by avidin-HRP in the presence of TMB (3,3',5,5'-tetramethylbenzidine) to generate a quantitative electrochemical signal. The time range for the entire protocol is ∼1 d, whereas the detection process takes ∼30 min to 3 h.

Programmable engineering of a biosensing interface with tetrahedral DNA nanostructures for ultrasensitive DNA detection.

Self-assembled DNA nanostructures with precise sizes allow a programmable "soft lithography" approach to engineer the interface of electrochemical DNA sensors. By using millimeter-sized gold electrodes modified with several types of tetrahedral DNA nanostructures (TDNs) of different sizes, both the kinetics and thermodynamics of DNA hybridization were profoundly affected. Because each DNA probe is anchored on an individual TDN, its lateral spacing and interactions are finely tuned by the TDN size. By simply varying the size of the TDNs, the hybridization time was decreased and the hybridization efficiency was increased. More significantly, the detection limit for DNA detection was tuned over four orders of magnitude with differentially nanostructured electrodes, and achieved attomolar sensitivity with polymeric enzyme amplification.

Multivalent capture and detection of cancer cells with DNA nanostructured biosensors and multibranched hybridization chain reaction amplification.

Sensitive detection of cancer cells plays a critically important role in the early detection of cancer and cancer metastasis. However, because circulating tumor cells are extremely rare in peripheral blood, the detection of cancer cells with high analytical sensitivity and specificity remains challenging. Here, we have demonstrated a simple, sensitive and specific detection of cancer cells with the detection sensitivity of four cancer cells, which is lower than the cutoff value with respect to correlation with survival outcomes as well as predictive of metastatic disease in clinical diagnostics. We re-engineered the hybridization chain reaction (HCR) to multibranched HCR (mHCR) that can produce long products with multiple biotins for signal amplification and multiple branched arms for multivalent binding. The capturing gold surface is modified with DNA tetrahedral probes, which provide superior hybridization conditions for the multivalent binding. The synergetic effect of mHCR amplification and multivalent binding lead to the high sensitivity of our detection platform.

Ultrasensitive electrochemical detection of prostate-specific antigen by using antibodies anchored on a DNA nanostructural scaffold.

The high occurrence of prostate cancer in men makes the prostate-specific antigen (PSA) screening test really important. More importantly, the recurrence rate after radical prostatectomy is high, whereas the traditional PSA immunoassay does not possess the sufficient high sensitivity for post-treatment PSA detection. In these assays, uncontrolled and random orientation of capture antibodies on the surface largely reduces their activity. Here, by exploiting the rapidly emerging DNA nanotechnology, we developed a DNA nanostructure based scaffold to precisely control the assembly of antibody monolayer. We demonstrated that the detection sensitivity was critically dependent on the nanoscale-spacing (nanospacing) of immobilized antibodies. In addition to the controlled assembly, we further amplified the sensing signal by using the gold nanoparticles, resulting in extremely high sensitivity and a low detection limit of 1 pg/mL. To test the real-world applicability of our nanoengineered electrochemical sensor, we evaluated the performance with 11 patients' serum samples and obtained consistent results with the "gold-standard" assays.

One-pot protocol for bimetallic Pt/Cu hexapod concave nanocrystals with enhanced electrocatalytic activity.

Nanomaterials with particular nanostructures which usually possess special properties always attract considerable attention. A novel bimetallic Pt/Cu hexapod nanostructure was prepared by a facile one-pot strategy. The formation mechanism was investigated by the time sequential evolution experiments and the hexapod concave nanostructures originated from the Pt/Cu rhombic dodecahedron. Further electrochemical measurements indicated the bimetallic Pt/Cu hexapod concave nanocrystals showed enhanced catalytic activities. It is believed that these novel nanostuctures would open up new opportunities for catalytic applications.