A molecular paradigm: "Plug-and-play" chemical sensing and crypto-steganography based on molecular recognition and selective response.

Inspired by information processing and communication in nature based on molecular recognition and structural diversity, ongoing efforts aim to development of artificial molecular or nano-systems for sensing, logic computing, and even data storage and safety. However, due to their preparation/functionalization shortcomings (laborious and time-consuming), poor flexibility and compatibility, and limited paradigm, it is still a big challenge whether simple molecules can be used to achieve comprehensive and universal applications from sensing to information storage and protection. Herein, we for the first time demonstrated a molecular paradigm-computer-like "basic input output system (BIOS)" which can realize "plug-and-play" sensing, information encoding, molecular cryptography, and steganography based on a simple artificial molecule (p-nitrophenol, PNP). Based on its molecular recognition and inherent chemical identities, PNP was utilized for colorimetric detection of multiple metal ions (Hg2+, Fe3+, Al3+, Cr3+) and distinguishing their valence (like Cr3+/Cr6+, Fe3+/Fe2+). Interestingly, PNP can achieve the "plug-and-play" fluorescent expansion of detection channels by directly mixing with fluorescent molecules, indicating that PNP molecule can be served as a molecular BIOS with flexibility and compatibility. Impressively, the selectivity embedded in PNP-based BIOS sensing system can be developed as molecular-level double cryptographic steganography to encode, encrypt and hide specific information (like the content of classical literature). This research not only provides a basic idea for building a molecular paradigm with "plug-and-play" flexibility and compatibility, but also provides ideas for the use of molecular sensing and informatization to open up the digitalization of the molecular world.

Microwave-Assisted Synthesis of Silver Nanoparticles for Multimode Colorimetric Sensing of Multiplex Metal Ions and Molecular Informatization Applications.

Plasmonic materials have been widely used in chemo/biosensing and biomedicine. However, little attention has been paid to the application of plasmonic materials in terms of the transition from molecular sensing to molecular informatization. Herein, we demonstrated that silver nanoparticles (AgNPs) prepared through facile and rapid microwave heating have multimode colorimetric sensing capabilities to different metal ions (Cr3+, Hg2+, and Ni2+), which can be further transformed into interesting and powerful molecular information technology (massively parallel molecular logic computing and molecular information protection). The prepared AgNPs can quantitatively and sensitively detect Cr3+ and Hg2+ in actual water samples. The AgNPs' multimode-guided multianalyte sensing processing was further investigated to construct a series of basic logic gates and advanced cascaded logic circuits by considering the analytes as the inputs and the colorimetric signals (like color, absorbance, wavelength shift) as the outputs. Moreover, the selective responses and molecular logic computing ability of AgNPs were also utilized to develop molecular cryptosteganography for encrypting and hiding some specific information, which proves that the molecular world and the information world are interconnected and use each other. This research not only opens the door for the transition from molecular sensing to molecular informatization but also provides an excellent opportunity for the construction of the "metaverse" of the molecular world.

Matter, energy and information network of a graphene-peptide-based fluorescent sensing system for molecular logic computing, detection and imaging of cancer stem cell marker CD133 in cells and tumor tissues.

Tumorigenesis, metastasis, and the recurrence of cancer, which may result from the abnormal presence or activation of cancer stem cells (CSCs), are involved in disorders of exchanged matter (biomarkers), energy and information in living organisms. Rapid and sensitive detection and imaging of CSC biomarkers (such as CD133) are helpful for early diagnosis and therapeutic evaluation of tumors. Recently, a preliminary exploration of a few affinity molecules (like peptide-based probes) has just begun for chemical measurements and imaging of CSC biomarker CD133. However, a comprehensive analysis of the matter, energy and information in an artificial molecular system has not been demonstrated and applied to biosensing and disease diagnosis. In this study, a graphene-peptide-based fluorescent sensing system was constructed by utilizing a graphene oxide platform and a CD133-specific recognition peptide and comprehensively analysed with respect to matter (molecular events), energy (fluorescence) and information flow. The molecular event interaction networks in this system were further used to perform molecular logic computing, for the sensitive detection of CSC marker CD133 (with a linear range from 0 to 630 nM and a detection limit of 7.91 nM), and for an application involving targeting the imaging of cells and tumor tissues that highly express CD133 (with a detection limit of 1.1 × 103 cells per mL for CT26 CSCs). The present report will provide more opportunities for the development and design of molecular-level intelligent complex systems and will probably promote the development of artificial intelligent sensing and treatment systems, a molecular-level "Internet of Things", and artificial life.

Graphene-Based Steganographically Aptasensing System for Information Computing, Encryption and Hiding, Fluorescence Sensing and in Vivo Imaging of Fish Pathogens.

Inspired by information processing and communication of life based on complex molecular interactions, some artificial (bio)chemical systems have been developed for applications in molecular information processing or chemo/biosensing and imaging. However, little attention has been paid to simultaneously and comprehensively utilize the information computing, encoding, and molecular recognition capabilities of molecular-level systems (such as DNA-based systems) for multifunctional applications. Herein, a graphene-based steganographically aptasensing system was constructed for multifunctional application, which relies on specific molecular recognition and information encoding abilities of DNA aptamers ( Aeromonas hydrophila and Edwardsiella tarda-binding aptamers as models) and the selective adsorption and fluorescence quenching capacities of graphene oxide (GO). Although graphene-DNA systems have been widely used in biosensors and diagnostics, our proposed graphene-based aptasensing system can not only be utilized for fluorescence sensing and in vivo imaging of fish pathogens ( A. hydrophila and E. tarda), but can also function as a molecular-level logic computing system where the combination of matters (specific molecules or materials) as inputs produces the resulting product (matter level) or fluorescence (energy level) changes as two outputs. More importantly and interestingly, our graphene-based steganographically aptasensing system can also serve as a generally doubly cryptographic and steganographic system for sending different secret messages by using pathogen-binding DNA aptamers as information carriers, GO as a cover, and a pair of keys, that is, target pathogen as a public key, the encryption key used to encode or decode a message in DNA as a private key. Our study not only provides a novel nanobiosensing assay for rapid and effective sensing and in vivo imaging of fish pathogens, but also demonstrates a prototype of (bio)molecular steganography as an important and interesting extension direction of molecular information technology, which is helpful in probably promoting the development of multifunctional molecular-level devices or machines.

The Boolean logic tree of molecular self-assembly system based on cobalt oxyhydroxide nanoflakes for three-state logic computation, sensing and imaging of pyrophosphate in living cells and in vivo.

Sensing of pyrophosphate (PPi) is helpful to better understand many life processes and diagnose various early-stage diseases. However, many traditional reported methods based on artificial receptors for sensing of PPi exhibit some disadvantages including difficulties in designing appropriate binding sites and complicated multi-step assembly/functionalization. Thus, it is significantly important and a big challenge to know how to use a simple molecular self-assembly or an interaction system to solve the above-mentioned limits to achieve the quantitative analysis of specific substances in the system. Based on the natural connection and similarity (such as stimulus responsiveness) between sensing and logic computing, in this study, the Boolean logic tree of molecular self-assembly system based on the cobalt oxyhydroxide (CoOOH) nanoplatform is constructed and applied to organize and connect "plug and play" molecular events (fluorescent dye, acridine orange and anion, PPi). By using molecules as inputs and the corresponding fluorescence signal as the output, the CoOOH-based molecular self-assembly system can be programmed for three-input fluorescent Boolean logic computation, fluorescent three-state logic computation, detection of PPi (linear range from 50 to 6400 nM with a detection limit of 20 nM) and even for imaging in living cancer cells and in vivo (in systems such as Zebrafish and Carassius auratus). Our approach adds a new dimension for expanding molecular logic computing and sensing systems, which will not only provide more opportunities for developing novel logic computing paradigms, but also be helpful in promoting the development and applications of intelligent molecular computing and sensing systems.

Directly repurposing waste optical discs with prefabricated nanogrooves as a platform for investigation of cell-substrate interactions and guiding neuronal growth.

Due to rapid change in information technology, many consumer electronics become electronic waste which is the fastest-growing pollution problems worldwide. In fact, many discarded electronics with prefabricated micro/nanostructures may provide a good basis to fulfill special needs of other fields, such as tissue engineering, biosensors, and energy. Herein, to take waste optical discs as an example, we demonstrate that discarded electronics can be directly repurposed as highly anisotropic platforms for in vitro investigation of cell behaviors, such as cell adhesion, cell alignment, and cell-cell interactions. The PC12 cells cultured on biocompatible DVD polycarbonate layers with flat and grooved morphology show a distinct cell morphology, indicating the topographical cue of nanogrooves plays a key role in guidance of neurites growth. By further monitoring cell morphology and alignment of PC12 cells cultured on the DVD nanogrooves at different differentiation times, we find that cell contact interaction with nanotopographies is dynamically adjustable with differentiation time from initial disorder to final order. This study adds a new dimension to not only solving the problems of supply of materials and fabrication of nanopatterns in neural tissue engineering, but may also offering a new promising way of waste minimization or reuse for environmental protection.

Highly Tunable and Scalable Fabrication of 3D Flexible Graphene Micropatterns for Directing Cell Alignment.

Patterning graphene allows to precisely tune its properties to manufacture flexible functional materials or miniaturized devices for electronic and biomedical applications. However, conventional lithographic techniques are cumbersome for scalable production of time- and cost-effective graphene patterns, thus greatly impeding their practical applications. Here, we present a simple scalable fabrication of wafer-scale three-dimensional (3D) graphene micropatterns by direct laser tuning graphene oxide reduction and expansion using a LightScribe DVD writer. This one-step laser-scribing process can produce custom-made 3D graphene patterns on the surface of a disk with dimensions ranging from microscale up to decimeter scale in about 20 min. Through control over laser-scribing parameters, the resulting various 3D graphene patterns are exploited as scaffolds for controlling cell alignment. The 3D graphene patterns demonstrate their potential to biomedical applications, beyond the fields of electronics and photonics, which will allow to incorporate flexible graphene patterns for 3D cell or tissue culture to promote tissue engineering and drug testing applications.

Boolean Logic Tree of Label-Free Dual-Signal Electrochemical Aptasensor System for Biosensing, Three-State Logic Computation, and Keypad Lock Security Operation.

The most serious and yet unsolved problems of molecular logic computing consist in how to connect molecular events in complex systems into a usable device with specific functions and how to selectively control branchy logic processes from the cascading logic systems. This report demonstrates that a Boolean logic tree is utilized to organize and connect "plug and play" chemical events DNA, nanomaterials, organic dye, biomolecule, and denaturant for developing the dual-signal electrochemical evolution aptasensor system with good resettability for amplification detection of thrombin, controllable and selectable three-state logic computation, and keypad lock security operation. The aptasensor system combines the merits of DNA-functionalized nanoamplification architecture and simple dual-signal electroactive dye brilliant cresyl blue for sensitive and selective detection of thrombin with a wide linear response range of 0.02-100 nM and a detection limit of 1.92 pM. By using these aforementioned chemical events as inputs and the differential pulse voltammetry current changes at different voltages as dual outputs, a resettable three-input biomolecular keypad lock based on sequential logic is established. Moreover, the first example of controllable and selectable three-state molecular logic computation with active-high and active-low logic functions can be implemented and allows the output ports to assume a high impediment or nothing (Z) state in addition to the 0 and 1 logic levels, effectively controlling subsequent branchy logic computation processes. Our approach is helpful in developing the advanced controllable and selectable logic computing and sensing system in large-scale integration circuits for application in biomedical engineering, intelligent sensing, and control.

Flexibility Analysis of Bacillus thuringiensis Cry1Aa.

OBJECTIVE: To investigate the flexibility and mobility of the Bacillus thuringiensis toxin Cry1Aa. METHODS: The graph theory-based program Constraint Network Analysis and normal mode-based program NMsim were used to analyze the global and local flexibility indices as well as the fluctuation of individual residues in detail. RESULTS: The decrease in Cry1Aa network rigidity with the increase of temperature was evident. Two phase transition points in which the Cry1Aa structure lost rigidity during the thermal simulation were identified. Two rigid clusters were found in domains I and II. Weak spots were found in C-terminal domain III. Several flexible regions were found in all three domains; the largest residue fluctuation was present in the apical loop2 of domain II. CONCLUSION: Although several flexible regions could be found in all the three domains, the most flexible regions were in the apical loops of domain II.

Residue 544 in domain III of the Bacillus thuringiensis Cry1Ac toxin is involved in protein structure stability.

A unique residue W544 in the beta18-beta19 loop of the Bacillus thuringiensis Cry1Ac toxin has been implicated in its toxicity. In this study, the effects of mutations at this residue on protein stability during protease treatment, UV irradiation, and preservation were examined. Residue 544 of Cry1Ac was involved in maintaining structural stability, and substitution of a polar group at this position was unfavorable to protein stability. One mutant, W544F, produced larger crystals and was more stable. This mutant showed greater resistance to UV radiation than the wild type Cry1Ac but retained equal toxicity. This is the first report showing that residue 544 in the Cry1Ac domain III plays a significant role in toxin structural stability. Our W544F mutant is a significant development in terms of field applications of Cry1Ac toxin.

The theoretical 3D structure of Bacillus thuringiensis Cry5Ba.

Cry5Ba is a delta-endotoxin produced by Bacillus thuringiensis PS86A1 NRRL B-18900. It is active against nematodes and has great potential for nematode control. Here, we predict the first theoretical model of the three-dimensional (3D) structure of a Cry5Ba toxin by homology modeling on the structure of the Cry1Aa toxin, which is specific to Lepidopteran insects. Cry5Ba resembles the previously reported Cry1Aa toxin structure in that they share a common 3D structure with three domains, but there are some distinctions, with the main differences being located in the loops of domain I. Cry5Ba exhibits a changeable extending conformation structure, and this special structure may also be involved in pore-forming and specificity determination. A fuller understanding of the 3D structure will be helpful in the design of mutagenesis experiments aimed at improving toxicity, and lead to a deep understanding of the mechanism of action of nematicidal toxins.

[Cloning and expression of the cry1Ac-tchiB fusion gene from Bacillus thuringinesis and Tobacco and its insecticidal synergistic effect].

A new fusion gene cry1Ac-tchiB was constructed to enhance the toxicity of crystal proteins, the cry1Ac gene of Bacillus thuringiensis strain 4.0718 was combined with a tchiB (deleted signal peptide and Enterokinase site sequence). In this process, the Enterokinase site sequence was inserted into the midst of these two genes. Then the combined fragment carrying the upstream promoter region and the downstream terminator region of cry1Ac gene were cloned into the shuttle vector pHT315. And after a series of enzyme digestions and subclonings two new expression vector pHUAccB6 and pHUAccB7 were obtained. The two vectors were transformed into B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant strain HAccB6 and HAccB7. The fusion gene was expressed and the expression product was detected by SDS-PAGE. The result indicated that the recombinant Cry1Ac-tchiB protein accumulated to the level of 61.38% of total bacterial proteins. Cry1Ac protein accumulated to the level of 42% of total bacterial proteins. Chitinase activities is 5.2 time more than that of the control strain. Under Atomic Force Microscopy and SEM of crystals protein, there were some bipy ramidal crystals with a size of 1.5 x 3.0 microm. Bioassay showed that the fusion crystals from recombinant strain HAccB6 and HAccB7 were high toxic against third-instar larvae of Helicourpa armigora with the LC50 (after 72h) value of 9.10 micro/mL and 11.34 micro/mL.The constructed fusion proteins had more toxicity than Cry1Ac crystal proteins. The study will enhances the toxicity of B. thuringiensis Cry toxins protein and makes a ground for constructing the fusion genes of B. thuringiensis cry gene and other foreign toxin genes and the recombinant strain with high toxicity.

[Analysis of insecticidal crystal proteins from Bacillus thuringiensis strain 4.0718 through two-dimensional gel electrophoresis and MALDI-TOF-mass spectrometry].

The present study analyses the insecticidal crystal proteins (ICPs) from Bacillus thuringiensis strain 4.0718 through two-dimensional electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). By comparing and optimizing the composition of lysis solution, the volume of sample loading and the protocol for isoelectric focusing, a well-focused 2-DE map with high resolution and reproducibility was achieved for the first time. And after an tryptic enzymolisis and a test of part of protein spots by means of MALDI-TOF-MS, the peptides mass fingerprint (PMF) was obtained and, by referring to Swiss-Prot, Cry1Ac and Cry2Aa contained in Bt 4.0718 were identified, with their molecular weights 134160 Da and 71097 Da respectively.

[Cloning and superexpression of cry1Ac gene from 20kb DNA associated with Bacillus thuringiensis Cry1A Crystal Protein].

The CrylA Crystal Protein from Bacillus thuringiensis is associated with DNA, but the role and sequences of these DNA molecules are unknown. CrylA bipyramidal crystals from B. thuringiensis strain 4.0718 was selectively dissolved and associated DNA was extracted from protoxin. The DNA was digested with Nde I to obtain 3 to 5 kb fragments and then the fragments were subcloned into pMD18-T vector, screening of recombinants were done by PCR-RFLP and sequencing. The ORF of cry1Ac gene was amplified by primers designed and then subcloned. The 3.5 kb BamH I and Sal I fragments of pMDX35 was inserted into the pET30a vector, giving 8.9 kb recombinant plasmid, pETX35. ETX35 strain were obtained by transformed pETX35 into B121 (DE3). A 141 kD fusion protein was superexpressed as inclusion bodies. Quantitative protein analysis indicated that the amount of 141 kD protein was above the level of 51.36% of total cellular protein. Plasmid pHTX42 constructed from shuttle vector pHT304 was transformed B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant HTX42. The recombinant protein was found with a molecular mass of 130 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scanning analysis indicated that the expressed protein accounted up to 79.28% of total cellular proteins and accumulated in the cells mounted up to 64.13% of cellular dry weight. Under Atomic Force Microscopy (AFM), typical bipyramidal crystals from HTX42 strain were found with a size of 1.2 microm x 2.0 microm. Bioassay showed that these inclusion bodies of ETX35 strain and crystals from HTX42 strain were highly toxic against the larvae of Plutella xylostella. On such a base, constructing insecticidal recombinant and analyzing the source, structure, and function of the 20 kb DNA can be further achieved.