Correction: Lonicera japonica Thunb. as a promising antibacterial agent for Bacillus cereus ATCC14579 based on network pharmacology, metabolomics, and in vitro experiments.

[This corrects the article DOI: 10.1039/D3RA00802A.].

Platelet-mimicking supramolecular nanomedicine with precisely integrated prodrugs for cascade amplification of synergistic chemotherapy.

Camptothecin (CPT) and cisplatin (Pt) have shown synergistic effects on a variety of cancers during preclinical and clinical studies. However, the ratio of the two drugs often could not be precisely regulated in different delivery systems, which hinders the desired synergistic effect. In addition, the low delivery efficiency of the two drugs to the tumor further impedes the ideal therapeutic outcomes. Herein, we report that a platelet-mimicking supramolecular nanomedicine (SN) could precisely control of the ratio of CPT and Pt with a high tumor accumulation rate for cascade amplification of synergistic chemotherapy. The SN was fabricated via the host-guest interaction between cucurbit[7]uril conjugated hyaluronic acid (HA-CB[7]) and adamantane (ADA) respectively functionalized CPT- and Pt-based prodrugs. The ratio of CPT and Pt in the SN could be facilely regulated by simply controlling the loading ratio, based on the strong binding affinity between CB[7] and ADA, and SN60 with 60% CPT and 40% Pt showed the highest synergistic effects on 4T1 cells. To improve the tumor accumulation efficiency of SN, 5,6-dimethylxanthenone-4-acetic acid (DMXAA, a tumor vasculature-disruptive agent) was loaded into the optimized SN and then coated with platelet membrane to yield platelet-mimicking supramolecular nanomedicine (D@SN-P). D@SN-P could first passively accumulate in tumors owing to the enhanced permeability and retention (EPR) effect after intravenous administration. The initially release of DMXAA from D@SN-P could induce tumor vascular disruption, and the resultant epithelial collagen exposure around the disrupted tumor vasculature provided a target for further recruitment of platelet-mimicking SN, leading to cascade amplification of tumor accumulation with synergistic chemotherapy. Hence, this platelet-mimicking supramolecular nanomedicine presents a universal supramolecular strategy to finely regulate the ratio of loaded pro-drugs, and improve the accumulation efficiency to amplify chemotherapy via platelet-mimics.

Lonicera japonica Thunb. as a promising antibacterial agent for Bacillus cereus ATCC14579 based on network pharmacology, metabolomics, and in vitro experiments.

Lonicera japonica Thunb. has attracted much attention for its treatment of bacterial and viral infectious diseases, while its active ingredients and potential mechanisms of action have not been fully elucidated. Here, we combined metabolomics, and network pharmacology to explore the molecular mechanism of Bacillus cereus ATCC14579 inhibition by Lonicera japonica Thunb. In vitro inhibition experiments showed that the Lonicera japonica Thunb.'s water extracts, ethanolic extract, luteolin, quercetin, and kaempferol strongly inhibited Bacillus cereus ATCC14579. In contrast, chlorogenic acid and macranthoidin B had no inhibitory effect on Bacillus cereus ATCC14579. Meanwhile, the minimum inhibitory concentrations of luteolin, quercetin, and kaempferol against Bacillus cereus ATCC14579 were 15.625 µg mL-1, 31.25 µg mL-1, and 15.625 µg mL-1. Based on the previous experimental basis, the metabolomic analysis showed the presence of 16 active ingredients in Lonicera japonica Thunb.'s water extracts and ethanol extracts, with differences in the luteolin, quercetin, and kaempferol contents between the water extracts and ethanol extracts. Network pharmacology studies indicated that fabZ, tig, glmU, secA, deoD, nagB, pgi, rpmB, recA, and upp were potential key targets. Active ingredients of Lonicera japonica Thunb. may exert their inhibitory effects by inhibiting ribosome assembly, the peptidoglycan biosynthesis process, and the phospholipid biosynthesis process of Bacillus cereus ATCC14579. An alkaline phosphatase activity assay, peptidoglycan concentration assay, and protein concentration assay showed that luteolin, quercetin, and kaempferol disrupted the Bacillus cereus ATCC14579 cell wall and cell membrane integrity. Transmission electron microscopy results showed significant changes in the morphology and ultrastructure of the cell wall and cell membrane of Bacillus cereus ATCC14579, further confirming the disruption of the cell wall and cell membrane integrity of Bacillus cereus ATCC14579 by luteolin, quercetin, and kaempferol. In conclusion, Lonicera japonica Thunb. can be used as a potential antibacterial agent for Bacillus cereus ATCC14579, which may exert its antibacterial activity by destroying the integrity of the cell wall and membrane.

Smartphone-based microplate reader for high-throughput quantitation of disease markers in serum.

Herein, a smartphone-based portable reader with integrated optics for standard microtiter plates (96 wells) has been designed and demonstrated for high-throughput quantitation of validated biomarkers in serum. The customized optical attachment was simply constructed with a convex lens and a light source, by which the transmitted light through a 96-well microtiter plate was converged for imaging with a smartphone, so that accurate and wide-range reading of the plate can be achieved. More importantly, relying on the digitized colorimetric analysis of the obtained images, concentrations of various biomarkers can be determined directly using the customized mobile app. A set of validated biomarkers for inflammation and infection, C-reactive protein (CRP), serum amyloid A (SAA), and procalcitonin (PCT) have been quantitated with this new system; both the response ranges and limits of detection meet the requirement of clinical tests. The consistency with the results obtained using a commercial microplate reader proves its reliability and precision, augments its potential as a point-of-care diagnostic device for on-site testing or resource-limited settings.

Quantitative pH Determination Based on the Dominant Wavelength Analysis of Commercial Test Strips.

The determination of pH values is essential in many chemical, medical, and environmental monitoring processes, which has been relying on conventional pH meters (glass electrodes) for quantitation and pH test strips for qualitative (or semi-quantitative) assessment. In this work, we demonstrate a smartphone-based pH determination technique, which performs digital image analysis of commercial test strips, particularly the determination of either the dominant wavelength (λd) or complementary wavelength (λc) of the color image. In conjunction with a 3D-printed optical accessory (with a surface light source and a macro lens), the quality of captured images have been warranted, and the quantitation accuracy of 0.05 pH units has been achieved. More importantly, the performance of this smartphone-based pH reading system (namely "Smart-pH-Reader") was validated using multiple real-world samples, as the results are consistent with those determined with a standard pH meter. The Smart-pH-Reader is envisioned to be a simple, portable, and accurate tool for pH determination in the fields of environmental monitoring, medical diagnosis, and beyond.

Development and Application of Mobile Apps for Molecular Sensing: A Review.

Modern smartphone-based sensing devices are generally standalone detection platforms that can transduce signals (via the built-in USB port, audio jack, or camera), perform analysis through mobile applications (apps), and display results on the screen/user interface. The advancement toward this ultimate form of on-site chemical analysis and point-of-care diagnosis is tied closely with the evolution of mobile technology. Previous reviews in the field mainly focused on the physical platforms while overlooking the role of mobile apps in such devices. There exist three general stages throughout the development: (1) early generation telemedicine, (2) mobile phone-assisted clinical diagnosis (without apps), and (3) mobile app-based sensing devices for various analytes. This review presents the key breakthroughs during each stage, recent development, remaining challenges, and future perspectives of the field. Representative examples, spanning from the pioneering point-of-care testing to the latest devices with integrated mobile apps, are classified by their sensing mechanisms. The review also discusses the scarcity of open-source apps dedicated to molecular sensing. With the introduction of more open-source and commercial apps, the mobile app-based detection system is anticipated to dominate point-of-care diagnosis and on-site molecular sensing in our opinion.

A Mobile Analytical Device for On-Site Quantitation of Anthocyanins in Fruit Beverages.

Anthocyanins are antioxidant and anti-inflammatory ingredients in various fruit beverages, for which their conservation and quantitation are important for the food industry. In this paper, we report a simple, portable device for accurate on-site determination of total monomeric anthocyanins in fruit beverages employing a Wi-Fi scanner coupled with a flexible microchip and a free mobile app. The detection principle is based on the pH-induced colorimetric reactions of anthocyanins performed in a specially designed microchip and validated with standard spectrophotometric measurements. The microchip with multiple testing vials was prepared with the benchtop molding method with a common PDMS elastomer and a transparency film; the photo of the scanned microchip is wirelessly sent to a smartphone and the RGB values of individual reaction vials in the microchip are analyzed with a free mobile app to determine the corresponding concentrations. It was demonstrated that the quantitation performance of this POCT device is comparable with conventional spectrophotometry in the determination of total anthocyanins in both standard solutions and fruit beverages.

A WiFi scanner in conjunction with disposable multiplex paper assay for the quantitation of disease markers in blood plasma.

Herein we report a quantitative, multiplex assay for disease markers in plasma based on an integrated setup of a portable scanner and a disposable paper-based analytical device (PAD). The quantitative analysis relies on the digital colorimetric reading of the three-layer PAD with 30 assay sites for performing respective chromogenic reactions for plasma uric acid, glucose, and triglyceride, which are considered as important risk factors for cardiovascular diseases. A portable scanner with WiFi transmission capability was used to produce high-quality color images of the PADs and wirelessly transfer them to a smartphone or other mobile devices for data processing. The concentrations of biomarkers in both standard solutions and plasma samples can be directly obtained using a custom-designed smartphone app that is also capable of constructing calibration curves. The detection limits of uric acid, glucose, and triglyceride were determined to be 0.50 mg/dL, 0.84 mmol/L, and 14 mg/dL, respectively, which are below the normal limits and adequate for clinical validation. Owing to the distinct advantages-simple, portable, and cost-effective-this mobile assay protocol can be used for point-of-care (POC) settings or resource-limited situations, and potentially for the diagnosis and prevention of infectious diseases.

Functional and versatile superhydrophobic coatings via stoichiometric silanization.

Superhydrophobic coatings have tremendous potential for applications in different fields and have been achieved commonly by increasing nanoscale roughness and lowering surface tension. Limited by the availability of either ideal nano-structural templates or simple fabrication procedures, the search of superhydrophobic coatings that are easy to manufacture and are robust in real-life applications remains challenging for both academia and industry. Herein, we report an unconventional protocol based on a single-step, stoichiometrically controlled reaction of long-chain organosilanes with water, which creates micro- to nano-scale hierarchical siloxane aggregates dispersible in industrial solvents (as the coating mixture). Excellent superhydrophobicity (ultrahigh water contact angle >170° and ultralow sliding angle <1°) has been attained on solid materials of various compositions and dimensions, by simply dipping into or spraying with the coating mixture. It has been demonstrated that these complete waterproof coatings hold excellent properties in terms of cost, scalability, robustness, and particularly the capability of encapsulating other functional materials (e.g. luminescent dyes).

A Long and Reversibly Self-Assembling 1D DNA Nanostructure Built from Triplex and Quadruplex Hybrid Tiles.

We report a new DNA nanostructure, an extended 1-dimensional composite built for the first time out of structurally robust yet conveniently disassembled DNA triple helices, interspersed with short stretches of G-quadruplexes. These "TQ Hybrid" 1-dimensional nanostructures require potassium ions and modestly acidic pH for their formation and are easily disassembled by changes to either of these requirements. We initially prepared and characterized a "monomeric" TQ Hybrid tile; followed by "sticky" TQs tiles, incorporating unique guanine-only sticky ends, that enable efficient self-assembly via G-quartet formation of nanostructures >150 nm in length, as seen with atomic force microscopy and transmission electron microscopy. We anticipate that such DNA TQ Hybrid structures will find unique and varied application as communication modules within larger nanostructures, and as sensors, logic gates, as well as in other aspects of DNA nanotechnology.

A colorimetric immuno-microarray for the quantitation and direct visualization of illicit drugs in body fluids.

The design and testing of integrated colorimetric microarray immunochips (immuno-microarrays) are reported for the quantitation and direct visual determination of multiple illicit drugs (e.g., morphine, cocaine and amphetamine) in body fluids. Such an immuno-microarray platform utilizes a competitive immunoassay format, which is based on silver staining for quantitative detection and multicolor staining for direct visualization (i.e., qualitative identification) of analytes present in the sample. Under optimized conditions, the dynamic response ranges of 3.7-1000, 1.1-300 and 1.5-300 ng mL-1 were achieved for amphetamine, cocaine, and morphine, respectively, which are wider towards low concentrations than those of standard enzyme-linked immunosorbent assay (ELISA) tests. The limits of detection (LODs) for morphine, cocaine, and amphetamine were determined to be 1.5 ± 0.1, 1.1 ± 0.1 and 3.7 ± 0.2 ng mL-1, respectively in oral fluids, which meet government regulations for law enforcement. The obvious advantages of multiplexing, simultaneous visual recognition, and accurate quantitation make the on-site detection feasible, confirming that such a colorimetric immuno-microarray holds promise for practical applications.

Measuring and Controlling the Local Environment of Surface-Bound DNA in Self-Assembled Monolayers on Gold When Prepared Using Potential-Assisted Deposition.

DNA self-assembled monolayers (SAMs) were prepared using potential-assisted deposition on clean gold single-crystal bead electrodes under a number of conditions (constant or square-wave potential perturbations in TRIS or phosphate immobilization buffers with and without Cl-). The local environment around the fluorophore-labeled DNA tethered to the electrode surface was characterized using in situ fluorescence microscopy during electrochemical measurements as a function of the underlying surface crystallography. Potential-assisted deposition from a TRIS buffer containing Cl- created DNA SAMs that were uniformly distributed on the surface with little preference to the underlying crystallography. A constant (+0.4 V/SCE) or a square-wave potential perturbation (+0.4 to -0.3 V/SCE, 50 Hz) resulted in similar DNA-modified surfaces in TRIS immobilization buffer. Deposition using a square-wave potential without Cl- resulted in lower DNA surface coverage. Despite this, the local environment around the DNA in the SAM appears to be densely packed. This implies the formation of clusters of densely packed DNA in the SAM. This effect was also demonstrated when depositing from a phosphate buffer. DNA clusters were significantly reduced when Cl- was present in the buffer. Clusters were most prevalent on the low-index plane surfaces (e.g., {111} and {100}) and less on the higher-index planes (e.g., {210} or {311}). A mechanism is proposed to rationalize the formation of DNA-clustered regions for deposition using a square-wave potential perturbation. The conditions for creating clusters of DNA in a SAM or for preventing these clusters from forming provide an approach for tailoring the surfaces used for biosensing.

CLICK-17, a DNA enzyme that harnesses ultra-low concentrations of either Cu+ or Cu2+ to catalyze the azide-alkyne 'click' reaction in water.

To enable the optimal, biocompatible and non-destructive application of the highly useful copper (Cu+)-mediated alkyne-azide 'click' cycloaddition in water, we have isolated and characterized a 79-nucleotide DNA enzyme or DNAzyme, 'CLICK-17', that harnesses as low as sub-micromolar Cu+; or, surprisingly, Cu2+ (without added reductants such as ascorbate) to catalyze conjugation between a variety of alkyne and azide substrates, including small molecules, proteins and nucleic acids. CLICK-17's Cu+ catalysis is orders of magnitude faster than that of either Cu+ alone or of Cu+ complexed to PERMUT-17, a sequence-permuted DNA isomer of CLICK-17. With the less toxic Cu2+, CLICK-17 attains rates comparable to Cu+, under conditions where both Cu2+ alone and Cu2+ complexed with a classic accelerating ligand, THPTA, are wholly inactive. Cyclic voltammetry shows that CLICK-17, unlike PERMUT-17, powerfully perturbs the Cu(II)/Cu(I) redox potential. CLICK-17 thus provides a unique, DNA-derived ligand environment for catalytic copper within its active site. As a bona fide Cu2+-driven enzyme, with potential for being evolved to accept only designated substrates, CLICK-17 and future variants promise the fast, safe, and substrate-specific catalysis of 'click' bioconjugations, potentially on the surfaces of living cells.

A versatile fluorometric in situ hybridization method for the quantitation of hairpin conformations in DNA self-assembled monolayers.

As the performance of hairpin DNA (hpDNA)-based biosensors is highly dependent on the yield of stem-loop (hairpin) conformations, we report herein a versatile fluorometric in situ hybridization protocol for examining hpDNA self-assembled monolayers (SAMs) on popularly used biochip substrates. Specifically, the ratio of fluorescence (FL) intensities of hpDNA SAMs (in an array format) before and after hybridization was adopted as the key parameter for performing such a determination. Upon confirming the existence of mixed and tunable DNA conformations in binary deposition solutions and efficient hybridization of the hairpin strands with the target DNA via gel electrophoresis assays, we tested the fluorometric protocol for determining the coverages of hpDNA in hpDNA/ssDNA SAMs prepared on gold; its accuracy was validated by Exonuclease I (Exo I)-assisted electrochemical quantitation. To further confirm its versatility, this FL protocol was adopted for quantifying hairpin conformations formed on glass and polycarbonate (PC) substrates. The molar ratios of surface-tethered hairpin conformations on the three different substrates were all found to be proportional to but less than those in the binary deposition solutions, and were dependent on the substrate morphology. The findings reported herein are beneficial for the construction of highly efficient DNA hairpin-based sensing surfaces, which essentially facilitates the creation of hpDNA-based biosensors with optimal detection performance.

Exonuclease I-Assisted General Strategy to Convert Aptamer-Based Electrochemical Biosensors from "Signal-Off" to "Signal-On".

In terms of how the signal varies in response to increased concentration of an analyte, sensors can be classified as either "signal-on" or "signal-off" format. While both types hold potentials to be sensitive, selective, and reusable, in many situations "signal-on" sensors are preferred for their low background signal and better selectivity. In this study, with the detection of lysozyme using its DNA aptamer as a trial system, for the first time we demonstrated that such an aptamer-based electrochemical biosensor can be converted from intrinsically "signal-off" to "signal-on" with the aid of a DNA exonuclease. The fact that the stepwise cleavage of antilysozyme aptamer catalyzed by Exonuclease I (Exo I) is entirely inhibited upon binding lysozyme leads to the selective removal of unbound DNA probes (thiolate anti-lysozyme DNA aptamer strands immobilized on gold electrode) upon the introduction of Exo I to the sensor. With the aid of electrostatically bound redox cations ([Ru(NH3)6]3+), we were able to quantitate the number of aptamer strands that are bound with lysozymes via conventional cyclic voltammetry (CV) measurements. We demonstrated that Exo I-assisted signal-on conversion protocol not only improves the sensing performance (10 times better limit of detection) but also promises a versatile strategy for DNA-based biosensor design, i.e., it can be readily adapted to other aptamer-protein binding systems (thrombin, as another example).

Indirect Competitive Immunoassay on a Blu-ray Disc for Digitized Quantitation of Food Toxins.

We report herein a Blu-ray disc technology enabled immunoassay (namely, assay-on-a-Blu-ray) protocol for the quantitation of food toxins. In particular, commercial Blu-ray discs (BDs) are activated as substrates to create indirect competitive immunoassays with the aid of microfluidic channel plates for the quantitation of aflatoxins; an unmodified Blu-ray drive is employed to read the digitized signal (error counts generated from gold/silver-particle-enhanced binding sites); and a free disc-quality control software is adapted to process the raw data. The performance of this BD-based digital detection platform has been tested for the quantitation of aflatoxin B1 (AFB1) in spiked corn samples and validated with standard high-performance liquid chromatography measurements. The detection limit attained is as low as 0.27 ppb with a dynamic response range up to 200 ppb, which meets the standards established by government agencies worldwide for food products. We truly believe that the application potential of such a BD-technology-based, portable device for multiplex on-site quantitative analysis of food products as well as environmental and biomedical samples in real time is unlimited.

Superhydrophobic Glass Microfiber Filter as Background-Free Substrate for Quantitative Fluorometric Assays.

We have discovered herein that commonly used laboratory glass microfiber filters can be functionalized as background-free superhydrophobic substrates for quantitative fluorometric assays. In particular, glass microfiber filters (Whatman GF/A) can be treated with low-concentration (20 mM) methyltrichlorosilane/toluene solution to be superhydrophobic (water contact angle >150°) in less than 5 min; the modified glass microfiber filter can be readily patterned with UV/ozone irradiation to create hydrophilic reaction zones on the otherwise superhydrophobic substrate. Compared with traditional cellulose filter paper, the glass microfiber filter has extremely low fluorescence background, which makes it an excellent substrate for preparing quantitative fluorometric assays. In conjunction with smartphone imaging and color analysis, we have showcased a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)-based fluorometric assay for copper quantitation on these patterned, superhydrophobic glass microfiber filter substrates. Both the limit of detection and linear response range are comparable with the standard spectrophotometric quantitation in solution and commercial copper detection kits, which augments the application potential of superhydrophobic glass microfiber filters as ideal (e.g., background-free) substrates for the preparation of multiplex microassays and other advanced microanalytical devices based on fluorescence readout.

Superhydrophobic Polydimethylsiloxane via Nanocontact Molding of Solvent Crystallized Polycarbonate: Optimized Fabrication, Mechanistic Investigation, and Application Potential.

Herein, we describe a benchtop protocol to create superhydrophobic polydimethylsiloxane (PDMS) via nanocontact molding of polycarbonate (PC) that was crystallized by controlled solvent treatment. The crystallized PC chains rearrange into a network of spherulites (spherical semicrystalline domains); the overall surface is rough on the micrometer-scale, while the spherulites themselves consist of nanoscale features. It was confirmed via conventional spectroscopic and high-resolution microscopic investigation that such hierarchical roughness is key to the development of superhydrophobic PC and the substantial enhancement upon PDMS molding. Thus, the prepared PDMS surface has excellent superhydrophobicity with an optimized contact angle of 172 ± 1° and a sliding angle of <1°, superior to those prepared from more elaborate techniques, such as plasma sputtering and laser etching. More importantly, the knowledge acquired regarding the structural transition and superhydrophobicity development would be beneficial to engineering and evaluating templates for many other polymeric nanostructures and functional surfaces.

Electrochemical Quantitation of Supramolecular Excipient@Drug Complexation: A General Assay Strategy Based on Competitive Host Binding with Surface-Immobilized Redox Guest.

The macrocyclic cucurbit[7]uril (CB[7]) host has exhibited great application potential as a pharmaceutical excipient due to its versatile abilities to modulate the chemical/physical properties of drug molecules (guests) and to control their in vivo delivery and release (upon complexation). The formation of stable CB[7]@drug complexes is the prerequisite for these promising applications; we report herein a general assay strategy to quantitate the complexation based on competitive binding with surface-immobilized redox guests in conjunction with conventional electrochemical techniques (e.g., cyclic voltammetry). Particularly, by incubating a mixture of CB[7] and a drug molecule with ferrocene (Fc)-terminated self-assembled monolayers (SAMs) on gold, the competitive host@guest binding between the CB[7]@drug complex formed in solution and the CB[7]@Fc complex formed on surface can be quantified with direct cyclic voltammetry measurements. On the basis of the known concentrations of CB[7]/drug and electrochemically determined surface densities of free/complexed Fc groups, the formation constant of CB[7]@drug complex can be determined. With several drug molecules as examples, we have demonstrated the capability of this method for quantitative studies of the formation of supramolecular excipient@drug complexes that are of interest in pharmaceutical and biomedical sciences. More importantly, this work promises a general assay strategy that allows electrochemical quantitation of a wide range of electro-inactive analytes based on the competitive supramolecular host@guest binding at redox-tagged molecular interfaces.

Blu-Ray Discs as Universal Biochip Substrates: Lithography-Free Surface Activation and Assay Patterning.

Blu-ray discs (BDs) are advantageous in comparison with other optical discs (compact discs and digital versatile discs) in terms of not only their storage capacity but also the high-quality materials fabricated from. We have recently discovered that the "Hard Coat" film of Verbatim BDs is in fact a unique type of polymeric substrates that can be readily activated and adapted for biochip fabrications. Particularly, the Hard Coat film peeled from BDs is optically transparent without any fluorescence background, which can be activated by treating with a common base (1.0 M NaOH) at a slightly elevated temperature (55 °C). The surface density of reactive carboxylic acid groups generated, 6.6 ± 0.7 × 10-9 mol/cm2, is much higher than that on polycarbonate upon UV/ozone irradiation (4.8 ± 0.2 × 10-10 mol/cm2). There are no significant physical damages to the substrate morphology, and the aging effect is minimal. More importantly, the BD substrate can be patterned using either cut-out filter paper masks or microfluidic channel plates; both are lithography-free, bench-top methods that facilitate the device fabrication in a common laboratory setting. With classical biotin-streptavidin binding and DNA hybridization arrays as trial systems, we have also demonstrated this new type of biochip substrates for quantitative assay applications.