Are Explicit Solvent Models More Accurate than Implicit Solvent Models? A Case Study on the Menschutkin Reaction

In this work, contemporary quantum mechanical (QM) implicit solvent models (SMD, SM12, and COSMO-RS) and a molecular mechanical (MM) explicit solvent model were used to predict the aqueous free energy barrier of a simple Menschutkin reaction (NH3 + CH3Cl). Surprisingly, the explicit solvent approach performed the worst, while the implicit solvent models yielded reasonably accurate values that are in accord with available experimental data. The origin of the large error in the explicit solvent model was due to the use of a fixed set of Lennard-Jones parameters during the free energy perturbation (FEP) calculations. Further analyses indicate that M06-2X/6-31+G­(d,p) yielded solute–solvent interaction energies that are in good agreement with benchmark DLPNO-CCSD­(T)/CBS values. When end-state MM to M06-2X/6-31+G­(d,p) corrections were added using FEP, it significantly improved the accuracy of the explicit solvent MM result and demonstrated that the accuracy of these models may be systematically improved with end-state corrections based on a validated QM level of theory

Assembly of Multiple DNA Components through Target Binding toward Homogeneous, Isothermally Amplified, and Specific Detection of Proteins

We describe a strategy of utilizing specific target binding to trigger assembly of three DNA components that are otherwise unable to spontaneously assemble with one another. This binding-induced DNA assembly forms a three-arm DNA junction, subsequently initiating nicking endonuclease-assisted isothermal fluorescence signal amplification. Real-time monitoring of fluorescence enables amplified detection of specific protein targets. The implementation of the strategy necessitates the simultaneous binding of a single target molecule with two affinity ligands each conjugated to a DNA motif. Simple alternation of affinity ligands enables different protein targets to induce the formation of the DNA junction and subsequent isothermal amplification. The use of the strategy allowed us to develop a sensitive assay for proteins with three appealing features: homogeneous analysis without the need for separation, isothermal amplification, and high specificity. Streptavidin was chosen as an initial target to establish and optimize the assay. Sensitivity of protein detection was improved by 1000-fold upon the application of isothermal amplification. A limit of detection of 10 pM was achieved for detection of prostate-specific antigen in buffer and diluted serum. The combination of its three appealing features makes the assay attractive for potential applications in molecular diagnosis, point-of-care testing, and on-site analysis

Binding-Induced Molecular Amplifier as a Universal Detection Platform for Biomolecules and Biomolecular Interaction

Techniques that detect multiple classes of biomolecules and biomolecular interactions from biological or patient samples are highly desirable for applications ranging from accurate disease diagnosis to deciphering comprehensive biological processes. Because of the large variations in target recognition, signal transduction, and instrumentation, it is technically challenging to generalize a single detection method to a diverse range of analytical targets. Herein, we introduce a binding-induced molecular amplifier (BIMA) strategy that translates a variety of biomolecules and biomolecular interactions into unified predesigned DNA barcode in homogeneous solutions. On the basis of a three-dimensional DNA-walking mechanism, BIMA not only translates various targets into a unified barcode but also amplifies the translation by generating multiple barcode molecules in response to a single input target molecule. Using this strategy, we have successfully expanded the uses of a simple toehold-mediated strand displacement beacon for the sensitive detection of multiple classes of targets, including nucleic acids, proteins, and protein–protein interactions

Enzyme-Powered Three-Dimensional DNA Nanomachine for DNA Walking, Payload Release, and Biosensing

Herein, we report a DNA nanomachine, built from a DNA-functionalized gold nanoparticle (DNA–AuNP), which moves a DNA walker along a three-dimensional (3-D) DNA–AuNP track and executes the task of releasing payloads. The movement of the DNA walker is powered by a nicking endonuclease that cleaves specific DNA substrates on the track. During the movement, each DNA walker cleaves multiple substrates, resulting in the rapid release of payloads (predesigned DNA sequences and their conjugates). The 3-D DNA nanomachine is highly efficient due to the high local effective concentrations of all DNA components that have been co-conjugated on the same AuNP. Moreover, the activity of the 3-D DNA nanomachine can be controlled by introducing a protecting DNA probe that can hybridize to or dehybridize from the DNA walker in a target-specific manner. This property allows us to tailor the DNA nanomachine into a DNA nanosensor that is able to achieve rapid, isothermal, and homogeneous signal amplification for specific nucleic acids in both buffer and a complicated biomatrix

Strand Displacement-Induced Enzyme-Free Amplification for Label-Free and Separation-Free Ultrasensitive Atomic Fluorescence Spectrometric Detection of Nucleic Acids and Proteins

In previous work, we have developed a simple strategy for a label-free and separation-free bioassay for target DNA and protein, with the limit of detection at the nM level only. Herein, taking advantage of atomic fluorescence spectrometric detection of metal ions and amplification of DNA, a label-free and separation-free ultrasensitive homogeneous DNA analytical platform for target DNA and protein detection was developed on the basis of an enzyme-free strand displacement signal amplification strategy for dramatically improved detectability. Using the T–Hg²⁺–T hairpin structure as the probe, the target DNA binds with HP (T–Hg²⁺–T hairpin structure) and released the Hg²⁺ first; then, the P4 (help DNA) hybridizes with target–P3 complex and free the target DNA, which is used to trigger another reaction cycle. The cycling use of the target amplifies the mercury atomic fluorescence intensity for ultrasensitive DNA detection. Moreover, the enzyme-free strand displacement signal amplification analytical system was further extended for protein detection by introducing an aptamer–P2 arched structure with thrombin as a model analyte. The current homogeneous strategy provides an ultrasensitive AFS detection of DNA and thrombin down to the 0.3 aM and 0.1 aM level, respectively, with a high selectivity. This strategy could be a promising unique alternative for nucleic acid and protein assay

Label-Free and Separation-Free Atomic Fluorescence Spectrometry-Based Bioassay: Sensitive Determination of Single-Strand DNA, Protein, and Double-Strand DNA

Based on selective and sensitive determination of Hg²⁺ released from mercury complex by cold vapor generation (CVG) atomic fluorescence spectrometry (AFS) using SnCl₂ as a reductant, a novel label-free and separation-free strategy was proposed for DNA and protein bioassay. To construct the DNA bioassay platform, an Hg²⁺-mediated molecular beacon (hairpin) without labeling but possessing several thymine (T) bases at both ends was employed as the probe. It is well-known that Hg²⁺ could trigger the formation of the hairpin structure through T–Hg²⁺–T connection. In the presence of a specific target, the hairpin structure could be broken and the captured Hg²⁺ was released. Interestingly, it was found that SnCl₂ could selectively reduce only free Hg²⁺ to Hg⁰ vapor in the presence of T–Hg²⁺–T complex, which could be separated from sample matrices for sensitive AFS detection. Three different types of analyte, namely, single-strand DNA (ssDNA), protein, and double-strand DNA (dsDNA), were investigated as the target analytes. Under the optimized conditions, this bioassay provided high sensitivity for ssDNA, protein, and dsDNA determination with the limits of detection as low as 0.2, 0.08, and 0.3 nM and the linear dynamic ranges of 10–150, 5–175, and 1–250 nM, respectively. The analytical performance for these analytes compares favorably with those by previously reported methods, demonstrating the potential usefulness and versatility of this new AFS-based bioassay. Moreover, the bioassay retains advantages of simplicity, cost-effectiveness, and sensitivity compared to most of the conventional methods

Heteromultivalent DNA Enhances the Assembly Yield of Hybrid Nanoparticles and Facilitates Dynamic Disassembly for Bioanalysis Using ICP–MS

To obtain enhanced physical and biological properties, various nanoparticles are typically assembled into hybrid nanoparticles through the binding of multiple homologous DNA strands to their complementary counterparts, commonly referred to as homomultivalent assembly. However, the poor binding affinity and limited controllability of homomultivalent disassembly restrict the assembly yield and dynamic functionality of the hybrid nanoparticles. To achieve a higher binding affinity and flexible assembly choice, we utilized the paired heteromultivalency DNA to construct hybrid nanoparticles and demonstrate their excellent assembly characteristics and dynamic applications. Specifically, through heteromultivalency, DNA-functionalized magnetic beads (MBs) and gold nanoparticles (AuNPs) were efficiently assembled. By utilizing ICP–MS, the assembly efficiency of AuNPs on MBs was directly monitored, enabling quantitative analysis and optimization of heteromultivalent binding events. As a result, the enhanced assembly yield is primarily attributed to the fact that heteromultivalency allows for the maximization of effective DNA probes on the surface of nanoparticles, eliminating steric hindrance interference. Subsequently, with external oligonucleotides as triggers, it was revealed that the disassembly mechanism of hybrid nanoparticles was initiated, which was based on an increased local concentration rather than toehold-mediated displacement of paired heteromultivalency DNA probes. Capitalizing on these features, an output platform was then established based on ICP–MS signals that several Boolean operations and analytical applications can be achieved by simply modifying the design sequences. The findings provide new insights into DNA biointerface interaction, with potential applications to complex logic operations and the construction of large DNA nanostructures

Characterization of IgG1 and IgM mAbs from EV-A71 infected children.

(A) numbers and percentages of EV-A71 neutralizing IgG1 mAbs, non-neutralizing EV-A71 binding IgG1 mAbs and non-binding IgG1 mAbs. (B) EV-A71 binding activity of 56 IgG1 mAbs from the five study subjects were determined using ELISA. (C) numbers and percentages of EV-A71 neutralizing IgM mAbs, non-neutralizing EV-A71 binding IgM mAbs and non-binding IgM mAbs. (D) EV-A71 binding activity of 56 IgM mAbs were determined by ELISA using mAb containing culture supernatants. EC50 (half maximal effective concentration) values for IgG1 mAbs and half-maximal binding dilution of IgM mAb containing culture supernatants were calculated using GraphPad Prism 7.</p

Lanthanide Encoded Logically Gated Micromachine for Simultaneous Detection of Nucleic Acids and Proteins by Elemental Mass Spectrometry

DNA-based logic computing potentially for analysis of biomarker inputs and generation of oligonucleotide signal outputs is of great interest to scientists in diverse areas. However, its practical use for sensing of multiple biomarkers is limited by the universality and robustness. Based on a proximity assay, a lanthanide encoded logically gated micromachine (LGM-Ln) was constructed in this work, which is capable of responding to multiplex inputs in biological matrices. Under the logic function controls triggered by inputs and a Boolean “AND” algorithm, it is followed by an amplified “ON” signal to indicate the analytes (inputs). In this logically gated sensing system, the whole computational process does not involve strand displacement in an intermolecular reaction, and a threshold-free design is employed to generate the 0 and 1 computation via intraparticle cleavage, which facilitates the computation units and makes the “computed values” more reliable. By simply altering the affinity ligands for inputs’ biorecognition, LGM-Ln can also be extended to multi-inputs mode and produce the robust lanthanide encoded outputs in the whole human serum for sensing nucleic acids (with the detection limit of 10 pM) and proteins (with the detection limit of 20 pM). Compared with a logically gated micromachine encoded with fluorophores, the LGM-Ln has higher resolution and no spectral overlaps for multiple inputs, thus holding great promise in multiplex analyses and clinical diagnosis

Demographic and clinical characteristics of the study subjects.

Demographic and clinical characteristics of the study subjects.</p