Simultaneous Quantification of Multiple Cancer Biomarkers in Blood Samples through DNA‐Assisted Nanopore Sensing

Protein biomarkers in blood have been widely used in the early diagnosis of disease. However, simultaneous detection of many biomarkers in a single sample remains challenging. Herein, we show that the combination of a sandwich assay and DNA‐assisted nanopore sensing could unambiguously identify and quantify several antigens in a mixture. We use five barcode DNAs to label different gold nanoparticles that can selectively bind specific antigens. After the completion of the sandwich assay, barcode DNAs are released and subject to nanopore translocation tests. The distinct current signatures generated by each barcode DNA allow simultaneous quantification of biomarkers at picomolar level in clinical samples. This approach would be very useful for accurate and multiplexed quantification of cancer‐associated biomarkers within a very small sample volume, which is critical for non‐invasive early diagnosis of cancer.     

A Universal Strategy for Aptamer‐Based Nanopore Sensing through Host–Guest Interactions inside α‐Hemolysin

Nanopore emerged as a powerful single‐molecule technique over the past two decades, and has shown applications in the stochastic sensing and biophysical studies of individual molecules. Here, we report a versatile strategy for nanopore sensing by employing the combination of aptamers and host–guest interactions. An aptamer is first hybridized with a DNA probe which is modified with a ferrocene?cucurbit[7]uril complex. The presence of analytes causes the aptamer–probe duplex to unwind and release the DNA probe which can quantitatively produce signature current events when translocated through an α‐hemolysin nanopore. The integrated use of magnetic beads can further lower the detection limit by approximately two to three orders of magnitude. Because aptamers have shown robust binding affinities with a wide variety of target molecules, our proposed strategy should be universally applicable for sensing different types of analytes with nanopore sensors.     

Impedance‐Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

In this paper, we present an impedance‐based DNA biosensor using thionine intercalation to amplify DNA hybridization signal. Beacon single‐stranded DNA (ssDNA) probe and mercaptoacetic acid were self‐assembled onto a Au electrode by forming Au? S bonds. These beacon ssDNAs were hybridized with the complementary sequences around the loop structure. Then thionine was intercalated into the double‐stranded DNA (dsDNA) immobilized on the Au electrode surface. Due to the neutralization of the negative charges of dsDNA by the intercalated thionine, the electronic transfer resistance (R_et) of the DNA modified Au electrode was significantly diminished. Herein, the decreased value of R_et resulted from the thionine intercalating into dsDNA was employed as the hybridization signal. SDS was used to reduce the unspecific adsorption between ssDNA and thionine. Several experimental conditions, including the surface coverage of ssDNA probe on Au electrode, the hybridization temperature and time were all optimized. Moreover, the hybridization reactions of the unstructured linear ssDNA probe and the structured beacon ssDNA probe with their complementary sequences were compared in this work. The sensitivity of the presented DNA biosensor highlighted that the intercalation of thionine into dsDNA was an efficient approach to amplify the hybridization signal using impedance detection technique. Additionally, in this DNA biosensing protocol, beacon ssDNA has a good ability to distinguish target DNA sequences. This results in a higher specificity than using traditional unstructured DNA probe.     

A Label-free and Functional Fluorescent Oligonucleotide Probe Based on a G-Quadruplex Molecular Beacon for the Detection of Kanamycin

A label-free and turn-off fluorescent method for the quantitative detection of kanamycin based on a functional molecular beacon was developed. The molecular beacon consists of two hairpin structures with a split G-rich oligonucleotide in the middle. The kanamycin's aptamer formed the loops portion for recognizing kanamycin, and the G-quadruplex bound by Thioflavin T(ThT) was employed as the reporter. In the absence of target, the molecular beacon folded into double stem-loops and the splited G-rich oligonucleotid came close to form a G-quadruplex. When ThT bound to the G-quadruplex, the fluorescence intensity of the solution increased. Upon the addition of kanamycin, the function between kanamycin and aptamer unfolded the hairpin and disassembled the G-quadraplex structure, resulting in a significant decrease in the fluorescence intensity. A good linear relationship ranging from 0.7 nmol/L to 10 nmol/L was achieved and the limit of detection was 0.37 nmol/L. Besides, it could efficiently recognize kanamycin in real samples.     

Substrate-Specific Amino Acid Sensing Using a Molecular d/ l-Cysteine Probe for Comprehensive Stereochemical Analysis in Aqueous Solution

The appearance of d -amino acids in mammals and humans has important implications in the life sciences. d /l -Amino acid mixtures play a key role in human physiology and pathology; thus, the introduction of artificial receptors for the real-time quantification of both the concentration and d /l composition of amino acids is very promising for the study of biological processes and for the diagnosis and treatment of diseases. We now report a sensing assay that is compatible with aqueous solutions and allows fast determination of the absolute configuration, enantiomeric composition, and overall amount of cysteine at micromolar concentrations. The method relies on fast UV and CD measurements, which provide accurate stereochemical information on samples covering a wide concentration range and drastically different d /l -cysteine ratios in simulated body fluids. Competition experiments show that other amino acids and biothiols do not interfere with the cysteine-targeted sensing.     

A Telomerase‐Specific Doxorubicin‐Releasing Molecular Beacon for Cancer Theranostics

A molecular beacon‐based drug delivery system was designed for both detection of telomerase activity in living cells and telomerase‐triggered drug release for precise cancer treatment. This system is composed of a gold nanoparticle core densely packed with FITC‐labeled hairpin DNA sequences hybridized with telomerase primers. Molecules of the anticancer drug doxorubicin were intercalated into the stem region of the DNA sequence. The presence of telomerase will elongate the primers, leading to inner chain substitution followed by the release of the FITC fluorescence and the trapped doxorubicin. This molecular beacon could specifically distinguish tumor cells and normal cells based on telomerase activity, precisely release doxorubicin in response to telomerase activity in the tumor cells, and prevent toxicity to normal organs.     

Activity‐Based Sensing: A Synthetic Methods Approach for Selective Molecular Imaging and Beyond

Emerging from the origins of supramolecular chemistry and the development of selective chemical receptors that rely on lock‐and‐key binding, activity‐based sensing (ABS)—which utilizes molecular reactivity rather than molecular recognition for analyte detection—has rapidly grown into a distinct field to investigate the production and regulation of chemical species that mediate biological signaling and stress pathways, particularly metal ions and small molecules. Chemical reactions exploit the diverse chemical reactivity of biological species to enable the development of selective and sensitive synthetic methods to decipher their contributions within complex living environments. The broad utility of this reaction‐driven approach facilitates application to imaging platforms ranging from fluorescence, luminescence, photoacoustic, magnetic resonance, and positron emission tomography modalities. ABS methods are also being expanded to other fields, such as drug and materials discovery.     

Redox‐Triggered Chirality Switching and Guest‐Capture/Release with a Pillar[6]arene‐Based Molecular Universal Joint

A chiral electrochemically responsive molecular universal joint (EMUJ) was synthesized by fusing a macrocyclic pillar[6]arene (P[6]) to a ferrocene‐based side ring. A single crystal of an enantiopure EMUJ was successfully obtained, which allowed, for the first time, the definitive correlation between the absolute configuration and the circular dichroism spectrum of a P[6] derivative to be determined. The self‐inclusion and self‐exclusion conformational change of the EMUJ led to a chiroptical inversion of the P[6] moiety, which could be manipulated by both solvents and changes in temperature. The EMUJ also displayed a unique redox‐triggered reversible in/out conformational switching, corresponding to an occupation/voidance switching of the P[6] cavity, respectively. This phenomenon is an unprecedented electrochemical manipulation of the capture and release of guest molecules by supramolecular hosts.     

DNA‐Programmed Dynamic Assembly of Quantum Dots for Molecular Computation

Despite the widespread use of quantum dots (QDs) for biosensing and bioimaging, QD‐based bio‐interfaceable and reconfigurable molecular computing systems have not yet been realized. DNA‐programmed dynamic assembly of multi‐color QDs is presented for the construction of a new class of fluorescence resonance energy transfer (FRET)‐based QD computing systems. A complete set of seven elementary logic gates (OR, AND, NOR, NAND, INH, XOR, XNOR) are realized using a series of binary and ternary QD complexes operated by strand displacement reactions. The integration of different logic gates into a half‐adder circuit for molecular computation is also demonstrated. This strategy is quite versatile and straightforward for logical operations and would pave the way for QD‐biocomputing‐based intelligent molecular diagnostics.     

Next‐Generation Sequencing as Input for Chemometrics in Differential Sensing Routines

Differential sensing (DS) methods traditionally use spatially arrayed receptors and optical signals to create score plots from multivariate data which classify individual analytes or complex mixtures. Herein, a new approach is described, in which nucleic acid sequences and sequence counts are used as the multivariate data without the necessity of a spatial array. To demonstrate this approach to DS, previously selected aptamers, identified from the literature, were used as semi‐specific receptors, Next‐Gen DNA sequencing was used to generate data, and cell line differentiation was the test‐bed application. The study of a principal component analysis loading plot revealed cross‐reactivity between the aptamers. The technique generates high‐dimensionality score plots, and should be applicable to any mixture of complex and subtly different analytes for which nucleic acid‐based receptors exist.     

Azulene‐Based Macrocyclic Receptors for Recognition and Sensing of Phosphate Anions

Herein we report the synthesis and detailed studies of the anion‐binding properties of two 20‐membered macrocyclic tetramide receptors: one symmetrical, containing two identical azulene‐based bisamide units, the other a hybrid, containing a dipicolinic bisamide unit and an azulene‐based bisamide unit. Analysis of the crystal structures of the macrocyclic receptors revealed their preference for adopting similar well‐preorganized bent‐sheet conformations, both as free receptors and in their complexes with anions. Studies of the optical properties of both receptors revealed abilities to selectively sense phosphate anions (H₂PO₄^?, HP₂O₇^3?), allowing for naked‐eye detection of the presence of these guests in DMSO. Binding studies in solution confirmed that the receptors bind strongly to a series of anions even in highly demanding media, such as mixtures of DMSO with water or with methanol. Comparison of the anion affinity of linear analogues with that of the macrocyclic receptors evidenced the importance of macrocyclic topology. Quantitative analysis revealed that the macrocyclic receptors are selective for H₂PO₄^? over other anions. The affinity to H₂PO₄^? seen for the symmetrical receptor, containing two azulene‐based subunits, is much higher than for the hybrid macrocycle containing both the azulene‐based and pyridine‐derived subunits. This highlights that the azulene‐based building block serves efficiently as both a binding site and a structure‐preorganizing motif.     

Dissipative Synthetic DNA‐Based Receptors for the Transient Loading and Release of Molecular Cargo

Supramolecular chemistry is moving into a direction in which the composition of a chemical equilibrium is no longer determined by thermodynamics but by the efficiency with which kinetic states can be populated by energy consuming processes. Herein, we show that DNA is ideally suited for programming chemically fueled dissipative self‐assembly processes. Advantages of the DNA‐based systems presented in this study include a perfect control over the activation site for the chemical fuel in terms of selectivity and affinity, highly selective fuel consumption that occurs exclusively in the activated complex, and a high tolerance for the presence of waste products. Finally, it is shown that chemical fuels can be used to selectively activate different functions in a system of higher complexity embedded with multiple response pathways.     

A Chemodosimetric Probe Based on a Conjugated Oxidized Bis‐Indolyl System for Selective Naked‐Eye Sensing of Cyanide Ions in Water

A new bis‐indolyl‐based colorimetric probe has been synthesized. This allows a Michael‐type adduct formation for the detection of cyanide ions. The probe shows a remarkable color change from red to colorless upon addition of the cyanide ions in pure water. The cyanide ion reacts with the probe and removes the conjugation of the bis‐indolyl moiety of the probe with that of the 4‐substituted aromatic ring. This renders the probe colorless. The mechanism of the reaction of the probe with the cyanide ion was established by using ¹H and ¹³C NMR spectroscopy, mass spectrometry, and kinetic studies.     

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.