Gelatin nanocarriers assembled by a self-immolative cross-linker for targeted cancer therapy.

With a number of outstanding properties, gelatin is an ideal candidate for assembling nanoplatforms in biomedical applications. Generally, gelatin nanocarriers are cross-linked by aldehydes to improve their stability in water solution. However, aldehydes could cause multiple toxicities and their cross-linking products are uncontrollable. Here, we first used a self-immolative cross-linker to assemble gelatin nanocarriers for the controlled release of drugs and targeted cancer therapy. The cross-linker contains a disulphide bridge and two symmetrical succinimidyl-esters, endowing it with multiple functions: 1) to cross-link the gelatin nanocarriers and thus improve their stability in water; 2) to conjugate the drug and tumor-targeting ligands with nanocarriers through covalent linkage; 3) to redox-responsively degrade the nanocarriers through hydrolysis of disulphide bridge; and 4) to produce traceless drug molecules through self-immolative reaction. Good biocompatibility and controllable drug release were demonstrated by in vitro experiments. Both qualitative and quantitative analyses confirmed the intracellular uptake of the nanocarriers by using doxorubicin (DOX) as a drug model and phenylboronic acid (PBA) as the targeting ligand. In vivo results demonstrated high therapeutic efficiency and low toxic side effects of the DOX loaded nanocarriers against artificial liver tumors.

Secretory production and characterization of a highly effective chitosanase from Streptomyces coelicolor A3(2) M145 in Pichia pastoris.

In this study, a glycoside hydrolase family 46 chitosanase from Streptomyces coelicolor A3(2) M145 was firstly cloned and expressed in Pichia pastoris GS115 (P. pastoris GS115). The recombinant enzyme (CsnA) showed maximal activity at pH 6.0 and 65°C. Both thermal stability and pH stability of CsnA expressed in P. pastoris GS115 were significantly increased compared with homologous expression in Streptomyces coelicolor A3(2). A stable chitosanase activity of 725.7 ± 9.58 U mL-1 was obtained in fed-batch fermentation. It's the highest level of CsnA from Streptomyces coelicolor expressed in P. pastoris so far. The hydrolytic process of CsnA showed a time-dependent manner. Chitosan oligosaccharides (COSs) generated by CsnA showed antifungal activity against Fusarium oxysporum sp. cucumerinum (F. oxysporum sp. cucumerinum). The secreted expression and hydrolytic performance make the enzyme a desirable biocatalyst for industrial controllable production of chitooligosaccharides with specific degree of polymerization, which have potential to control fungi that cause important crop diseases.

No-cost ballpoint pen dispenser for lateral flow assays.

We have developed a no-cost, lightweight, human-powered dispenser using an empty ballpoint pen. Used in lateral flow assays, this dispenser restricts antibody deposition to narrow zones, allowing freehand drawing of test and control lines. The lines can be drawn in widths ranging from 0.15 to 1.00 mm. Naphthol green B, a compatible stain, was used to label antibody solutions and certify handwriting traces. Using human chorionic gonadotropin (HCG) as a model antigen, we demonstrated that the pen dispenser can imprint antibodies on nitrocellulose membranes without affecting their microstructure and chromatographic function. A lateral flow assay using the pen dispenser detected HCG at 0.1 µg/mL, comparable to the sensitivity of standard tests using traditional benchtop dispensers.

Reversible cross-linking of gelatin by a disulphide-containing bis-succinimide for tunable degradation and release.

Generally, gelatin was irreversibly cross-linked by chemical reagents to improve its water-resistance. However, few chemical reagents meet both the requirements of high cross-inking efficiency and tunable degradation. Here a reversible cross-linker, disulphide-containing bis-succinimide, was synthesized and used to control the cross-linking and degradation of edible gelatin film. Mixture of the gelatin and cross-linker for 120 min generated gelatin films that could preserve their morphology in 37 ℃ warm water for above 40 days. The gelatin film changed its microstructure from net to tightness after the cross-linking, thus facilitating the embedding of the targeted molecule into the gelatin material. The degradation of the cross-linked gelatin film and the release of its inclusion could be controlled by biocompatible glutathione. This work provides a good method for preparing modified gelatin with promising water-resistance, good biocompatibility, and tunable degradation for food/biomedical engineering applications.

Intrafibrillar mineralization of type I collagen by micelle-loaded amorphous calcium phosphate nanoparticles.

Mineralization of type I collagen fibrils is highly desired for artificial bone preparation and teeth repairing. Generally, amorphous calcium phosphate (ACP) combined with non-collagenous protein analogue (NCPA) were used for biomimetic remineralization of collagen fibrils. However, the ACP was likely to aggregate to form larger particles that could not infiltrate into the gaps of the collagen for intrafibrillar mineralization, and the poor storage stability of ACP has challenged its practical applications. To address this question, here we assembled ACP that was stabilized by carboxylated polyamidoamine (CPAMAM) at a pH of 6.5 to form dispersed nanoparticles of 25 nm in size, which was named as ACP/CPAMAM. The ACP/CPAMAM nanoparticles were further loaded into micelles composed of polysorbate and polyethylene glycol (PEG) to further improve their storage stability. The micelle-loaded ACP/CPAMAM particles could maintain their amorphous phase after storage for 12 months. During the mineralization of collagen fibrils, isopropanol (IPA) was introduced to dissolve the micelles and release the ACP/CPAMAM nanoparticles. By using micelle-loaded ACP/CPAMAM, good intrafibrillar mineralization of type I collagen was demonstrated. This work provides novel methods for preparing ACP nanoparticles with good storage stability and controllable release for intrafibrillar mineralization.

Bridge-DNA synthesis triggered by an allosteric aptamer for the colorimetric detection of pathogenic bacteria.

Rapid and sensitive quantification of pathogenic bacteria is highly desired for environmental health supervision and food safety control. Yet, the amplification and detection of bacteria with a concentration lower than 102 cfu mL-1 remains a great challenge. Here, we combined an allosteric aptamer (AAP) with a gold nanoparticle (AuNP) for assembling a bridge-DNA synthesis system (named as AuNP-BDS) to amplify the bacterial signals. The AAP and its paired primer (PP) were covalently linked to two different AuNPs, respectively: one named as AAP-AuNP and the other PP-AuNP. Upon recognition of the antigen from the pathogenic bacteria, AAP alters its conformation to initiate DNA synthesis on the AuNP surface. The DNA products from AAP-AuNP and PP-AuNP form bridges to each other through base pairing, resulting in the aggregation and colorimetric response of the AuNPs. By using E. coli O157:H7 as an example, the AuNP-BDS could quantify pathogenic bacteria in water with a concentration as low as 10 cfu mL-1 within 60 min and without any enrichment. The colorimetric response values of AuNP-BDS were found to be linearly related to the bacterial concentrations in the range of 10 to 103 cfu mL-1. Good practicability of the AuNP-BDS in quantifying E. coli O157:H7 from tap water, juices, and milks was demonstrated. The AuNP-BDS could be exploited to facilitate the rapid and sensitive quantification of pathogenic bacteria for food safety control.

Preparation, characteristics and cytotoxicity of green synthesized selenium nanoparticles using Paenibacillus motobuensis LY5201 isolated from the local specialty food of longevity area.

Selenium is an essential micronutrient element. For the extremely biotoxic of selenite, Selenium nanoparticles (SeNPs) is gaining increasing interest. In this work, a selenium-enriched strain with highly selenite-resistant (up to 173 mmol/L) was isolated from the local specialty food of longevity area and identified as Paenibacillus motobuensis (P. motobuensis) LY5201. Most of the SeNPs were accumulated extracellular. SeNPs were around spherical with a diameter of approximately 100 nm. The X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy showed that the purified SeNPs consisted of selenium and proteins. Our results suggested that P. motobuensis LY5201could be a suitable and robust biocatalyst for SeNPs synthesis. In addition, the cytotoxicity effect and the anti-invasive activity of SeNPs on the HepG2 showed an inhibitory effect on HepG2, indicating that SeNPs could be used as a potential anticancer drug.

On-bead DNA synthesis triggered by allosteric probe for detection of carcinoembryonic antigen.

Sensitive quantification of protein biomarkers is highly desired for clinical diagnosis and treatment. Yet, unlike DNA/RNA which can be greatly amplified by PCR/RT-PCR, the amplification and detection of trace amount of proteins remain a great challenge. Here, we combined allosteric probe (AP) with magnetic bead (MB) for assembling an on-bead DNA synthesis system (named as APMB) to amplify protein signals. The AP is designed and conjugated onto the MB, enabling the protein biomarker to be separated and enriched. Once recognizing the biomarker, the AP alters its conformation to initiate DNA synthesis on beads for primary signal amplification. During the DNA synthesis, biotin-dATPs are incorporated into the newly synthesized DNA strands. Then, the biotin-labeled DNA specifically captures streptavidin (STR)-conjugated horseradish peroxidase (HRP), which is used to catalyze a colorimetric reaction for secondary signal amplification. By using carcinoembryonic antigen (CEA) as a protein model, the APMB can quantify protein biomarkers of as low as 0.01 ng/mL. The response values measured by APMB are linearly related to the protein concentrations in the range 0.05 to 20 ng/mL. Clinical examination demonstrated good practicability of the APMB in quantifying serum protein biomarker. The on-bead DNA synthesis could be exploited to improve protein signal amplification, thus facilitating protein biomarker detection of low abundance for early diagnosis.

Using a safe and effective fixative to improve the immunofluorescence staining of bacteria.

The emerging and development of green chemistry has once again drawn the researchers' attention to eliminating the use and generation of hazardous materials. Here we report the use of a safe and effective fixative, chlorine dioxide (ClO2), instead of traditional hazardous fixatives for the cross-linking of cellular proteins to improve immunofluorescence staining of bacteria. The concentration of ClO2needed for 100% fixation is 50µg ml-1, which is much lower than that of traditional fixatives (1000-10000µg ml-1). The ClO2mediated cross-linking can preserve the integrity of bacterial cells and prevent cell loss through lysis. Meanwhile, lysozyme can permeabilize the bacterial cells, allowing the labelled antibodies to diffuse to their intracellular target molecules. By usingE. coliO157:H7/RP4 as a gram-negative bacteria model, immunofluorescence staining assays for both intracellular protein and surface polysaccharide were carried out to investigate the effect of ClO2fixation on the staining. The results demonstrated that ClO2fixation could prevent the target antigens from cracking off the bacteria without damage on the interaction between the antibodies and antigens (either for polysaccharide or protein). As a safe and effective fixative, ClO2has potential practical applications in immunofluorescence staining and fluorescencein situhybridization for single bacteria/cell analysis.

Assembly of "carrier free" enzymatic nano-reporters for improved ELISA.

Enzyme-linked immunosorbent assay (ELISA) is an economic and easy operation technique that has been widely used for the detection of protein in industry. However, the low loading capacity of the enzyme reporter has contributed to the low sensitivity of traditional ELISA, and the cross-linking procedures of the enzyme-labeled antibody in ELISA methods can lead to the inactivation of the enzyme, which will further decrease the sensitivity. To address this issue, herein we fabricated "carrier-free" nanoparticles to obtain a horseradish peroxidase (HRP) labelled reporter with a high HRP loading capacity. A disulphide-containing bis-N-hydroxy succinimide (NHS) crosslinker (NHS-SS-NHS) was used to control the link and release of traceless HRPs, thus without reduction of its enzymatic activity. The HRP nanoparticle (NanoHRP) was successfully applied for dot blotting and ELISA. When carcinoembryonic antigen (CEA) was used as a target, the detection limit of the NanoHRP-based ELISA was 0.005 ng mL-1, which was about 400 times more sensitive than traditional ELISA. A good correlation between the CEA concentrations and the response values measured by NanoHRP ELISA was obtained in the range of 0.005 to 1 ng mL-1. This concept could be exploited to improve ELISA tests, especially those requiring a high accuracy, to facilitate physicians in deciding the appropriate medical treatment.

Rapid quantification of pathogenic Salmonella Typhimurium and total bacteria in eggs by nano-flow cytometry.

Rapid quantification of pathogenic Salmonella Typhimurium (S. Typhimurium) and total bacteria in eggs is highly desired for food safety control. However, the complexity of egg matrix presents a significant challenge for sensitive detection of bacteria. In this study, a sample pretreatment protocol, including dilution, fat dissolution, protein degradation, filtration, and washing was developed to circumvent this challenge. A laboratory-built nano-flow cytometer (nFCM) that is hundreds of fold more sensitive than the conventional flow cytometer was employed to analyze individual bacteria upon nucleic acid and immunofluorescent staining. Eggs spiked with pathogenic S. Typhimurium and harmless Escherichia coli K12 (E. coli K12) were used as the model system to optimize the sample pretreatment protocol. S. Typhimurium and total bacteria in eggs can be quantified without cultural enrichment, and the whole process of sample pretreatment, staining, and instrument analysis can be accomplished within 1.5 h. The bacterial recovery rate upon sample pretreatment, detection limit, and dynamic range for S. Typhimurium in eggs were 92%, 2 × 103 cells/mL, and from 2 × 103 to 4 × 108 cells/mL, respectively. The as-developed approach can specifically distinguish S. Typhimurium from other bacteria and successful application to bacterial detection in eggs freshly purchased from supermarket and spoiled eggs upon inappropriate storage was demonstrated.

Label-Free Detection of Bacteria in Fruit Juice by Nano-Flow Cytometry.

Rapid quantification of microbial contamination in fruit juice is highly desired for food safety control. Yet, the complex sample matrix and the diversity of bacterial contaminants present a great challenge. Employing a laboratory-built nano-flow cytometer (nFCM), here we report the development of a label-free approach for the detection of bacteria population in fruit juice. The weak autofluorescence of bacterial cells was used as a hallmark for the identification of bacteria. The sample pretreatment protocol was optimized to reduce fluorescence background, lyse residual plant cells and debris, and attain a good recovery of bacteria from juice samples. It was demonstrated that the nFCM was able to enumerate individual bacteria of very weak autofluorescence, and a clear differentiation from residual juice particulates was achieved. For bacteria spiked in the orange juice, the recovery rate was around 95% and a linear correlation between nFCM analysis and plate counting was acquired in the range of 3 × 104 to 3 × 108 cfu/mL. The assay, including sample pretreatment and instrument analysis, can be accomplished within 1 h, which is far more efficient than plate counting. Using a 40 mL sample volume, the detection limit in apple juice was ∼102 cells/mL. The as-developed method was successfully applied to bacterial measurement of freshly made orange juice and apple juice purchased from grocery stores. We believe it could also have potential practical application in microbial control analysis of other juices and water.

Deciphering the Antitoxin-Regulated Bacterial Stress Response via Single-Cell Analysis.

Bacterial toxin-antitoxin (TA) systems, which are diverse and widespread among prokaryotes, are responsible for tolerance to drugs and environmental stresses. However, the low abundance of toxin and antitoxin proteins renders their quantitative measurement in single bacteria challenging. Employing a laboratory-built nano-flow cytometer (nFCM) to monitor a tetracysteine (TC)-tagged TA system labeled with the biarsenical dye FlAsH, we here report the development of a sensitive method that enables the detection of basal-level expression of antitoxin. Using the Escherichia coli MqsR/MqsA as a model TA system, we reveal for the first time that under its native promoter and in the absence of environmental stress, there exist two populations of bacteria with high or low levels of antitoxin MqsA. Under environmental stress, such as bile acid stress, heat shock, and amino acid starvation, the two populations of bacteria responded differently in terms of MqsA degradation and production. Subsequently, resumed production of MqsA after amino acid stress was observed for the first time. Taking advantage of the multiparameter capability of nFCM, bacterial growth rate and MqsA production were analyzed simultaneously. We found that under environmental stress, the response of bacterial growth was consistent with MqsA production but with an approximate 60 min lag. Overall, the results of the present study indicate that stochastic elevation of MqsA level facilitates bacterial survival, and the two populations with distinct phenotypes empower bacteria to deal with fluctuating environments. This analytical method will help researchers gain deeper insight into the heterogeneity and fundamental role of TA systems.

Flow Cytometric Analysis of Nanoscale Biological Particles and Organelles.

Analysis of nanoscale biological particles and organelles (BPOs) at the single-particle level is fundamental to the in-depth study of biosciences. Flow cytometry is a versatile technique that has been well-established for the analysis of eukaryotic cells, yet conventional flow cytometry can hardly meet the sensitivity requirement for nanoscale BPOs. Recent advances in high-sensitivity flow cytometry have made it possible to conduct precise, sensitive, and specific analyses of nanoscale BPOs, with exceptional benefits for bacteria, mitochondria, viruses, and extracellular vesicles (EVs). In this article, we discuss the significance, challenges, and efforts toward sensitivity enhancement, followed by the introduction of flow cytometric analysis of nanoscale BPOs. With the development of the nano-flow cytometer that can detect single viruses and EVs as small as 27 nm and 40 nm, respectively, more exciting applications in nanoscale BPO analysis can be envisioned.

Light-Scattering Sizing of Single Submicron Particles by High-Sensitivity Flow Cytometry.

Rapid and reliable size measurement of single submicron particles (100-1000 nm) is important for quality control of particulate matter, biomedical research, environmental study, and drug delivery system development. Though direct measurement of the elastically scattered light from individual submicron particles represents the simplest method for particle size measurement, the inadequate instrument sensitivity and complicated relationship between scattering intensity and particle size render it a great challenge. Combining the superior sensitivity of a laboratory-built high-sensitivity flow cytometer (HSFCM) in the side scattering (SSC) detection of single nanoparticles and the great efforts in synthesizing 38 highly monodisperse silica spheres ranging from 180 to 880 nm with small size intervals, here we report the first comprehensive comparison between the experimentally measured and Mie theory calculated intensities of light scattered by single submicron particles. Good agreements were observed for both the silica spheres and polystyrene beads at both the perpendicular and the parallel polarizations of the incident laser beam. Compared with perpendicular polarization, parallel polarization can resolve differently sized beads better due to the continuously increased scattering intensity with particle size. The predictive capability of the simple numerical model constructed in present work can be exploited to allow us to foresee scattering behavior on flow cytometers. More importantly, the linear correlation between the measured and the calculated scattering intensities enables us to develop a method that can measure the particle size of submicron particles with the precision and accuracy of Mie theory rather than a calibration curve fitted by several sparsely separated size reference standards. Comparable sizing resolution and accuracy to those of electron microscopy were demonstrated for Gram-positive bacteria Staphylococcus aureus. The as-developed method shows great potential in guiding the accurate size measurement of submicron particles.

Rapid quantification of live/dead lactic acid bacteria in probiotic products using high-sensitivity flow cytometry.

A laboratory-built high-sensitivity flow cytometer (HSFCM) was employed for the rapid and accurate detection of lactic acid bacteria (LAB) and their viability in probiotic products. LAB were stained with both the cell membrane-permeable SYTO 9 green-fluorescent nucleic acid stain and the red-fluorescent nucleic acid stain, propidium iodide, which penetrates only bacteria with compromised membranes. The side scatter and dual-color fluorescence signals of single bacteria were detected simultaneously by the HSFCM. Ultra-high temperature processing milk and skim milk spiked with Lactobacillus casei were used as the model systems for the optimization of sample pretreatment and staining. The viable LAB counts measured by the HSFCM were in good agreement with those of the plate count method, and the measured ratios between the live and dead LAB matched well with the theoretical ratios. The established method was successfully applied to the rapid quantification of live/dead LAB in yogurts and fermented milk beverages of different brands. Moreover, the concentration and viability status of LAB in ambient yogurt, a relatively new yet popular milk product in China, are also reported.

Immobilization of chlorine dioxide modified cells for uranium absorption.

There has been a trend towards the use of microorganisms to recover metals from industrial wastewater, for which various methods have been reported to be used to improve microorganism adsorption characteristics such as absorption capacity, tolerance and reusability. In present study, chlorine dioxide(ClO2), a high-efficiency, low toxicity and environment-benign disinfectant, was first reported to be used for microorganism surface modification. The chlorine dioxide modified cells demonstrated a 10.1% higher uranium adsorption capacity than control ones. FTIR analysis indicated that several cell surface groups are involved in the uranium adsorption and cell surface modification. The modified cells were further immobilized on a carboxymethylcellulose(CMC) matrix to improve their reusability. The cell-immobilized adsorbent could be employed either in a high concentration system to move vast UO2(2+) ions or in a low concentration system to purify UO2(2+) contaminated water thoroughly, and could be repeatedly used in multiple adsorption-desorption cycles with about 90% adsorption capacity maintained after seven cycles.

Laboratory investigation of reducing two algae from eutrophic water treated with light-shading plus aeration.

The occurrence of harmful algal bloom in water source poses a serious water safety problem to local water supply systems. In order to ensure the raw water quality, the feasibility of reducing harmful algae by light-shading plus aeration was investigated. The batch test showed that algal biomass reduced rapidly under light-shading condition, and the reduction efficiency was further increased when light-shading was accompanied by aeration. The continuous flow experiment showed that the algal reduction efficiency increased with the increase of residence time. At residence time of 5 d, when treated with light-shading plus aeration, algal biomass could be reduced by more than 65%, with raw water quality improved simultaneously. Furthermore, considering that some harmful algae such as Microcystis tend to float upwards under light-limited condition, an integrated light-shading system consisting of pre-separation process and light-shading plus aeration treatment was suggested to treat naturally high algal water. The result showed that pre-separation process could remove more than 40% of algal biomass, and the total reduction efficiency of the integrated system increased to above 80%.