A universal strategy of glyconanoparticle preparation using a bifunctional linker for lectin sensing and cell imaging.

Glyconanoparticles (G-NPs), biofunctional nanomaterials that can fully combine the unique properties of nanoparticles (NPs) with the bioactivities of carbohydrates, have become an appealing nanoplatform in analytical chemistry and biomedical research. However, there is currently a lack of an efficient and universal method for facile immobilization of reducing carbohydrates on NPs while maintaining their structure integrity, greatly limiting the preparation and application of G-NPs. Herein, a new and universal strategy for preparing carbohydrate-functionalized gold nanoclusters (Au NCs) was developed by using S-(3-(methoxyamino)propyl) thioacetate (MPTA) as a new bifunctional linker. MPTA with an N-methoxyamine group (-NHOMe) and a thioacetyl group (-SAc) was synthesized by a two-step strategy and then grafted onto Au NCs by an efficient click reaction. Subsequently, reducing carbohydrates could be readily immobilized onto MPTA-functionalized Au NCs (MPTA-Au NCs) by a reducing end ring-closure reaction under mild conditions. The obtained G-NPs showed average size of 1.9 ± 0.42 nm and strong fluorescence at 610 nm. Carbohydrates grafted on G-NPs still retained their structure integrity and specific recognition ability toward their receptor proteins. Notably, the affinity between G-NPs and proteins was increased by 1300 times compared with free carbohydrates with an association constant of (1.47 ± 0.356) × 106 M-1. The prepared fluorescent G-NPs were also successfully applied to lectin sensing and targeted breast cancer cell imaging with good performance. These results indicated that the intact immobilization of reducing carbohydrates (whether naturally or chemically accessed) on NPs could be easily achieved using MPTA, providing a simple, efficient, and universal strategy for G-NP preparation.

Hand-powered vacuum-driven microfluidic gradient generator for high-throughput antimicrobial susceptibility testing.

The growth of bacterial resistance to antimicrobials is a serious problem attracting much attention nowadays. To prevent the misuse and abuse of antimicrobials, it is important to carry out antibiotic susceptibility testing (AST) before clinical use. However, conventional AST methods are relatively laborious and time-consuming (18-24 h). Here, we present a hand-powered vacuum-driven microfluidic (HVM) device, in which a syringe is used as the only vacuum source for rapid generating concentration gradient of antibiotics in different chambers. The HVM device can be preassembled with various amounts of antibiotics, lyophilized, and stored for ready-to-use. Bacterial samples can be loaded into the HVM device through a simple suction step. With the assistance of Alamar Blue, the AST assay and the minimum inhibitory concentration (MIC) of different antibiotics can be investigated by comparing the growth results of bacteria in different culture chambers. In addition, a parallel HVM device was proposed, in which eight AST assays can be performed simultaneously. The results of MIC of three commonly used antibiotics against E. coli K-12 in our HVM device were consistent with those obtained by traditional method while the detection time was shortened to less than 8 h. We believe that our platform is high-throughput, cost-efficient, easy to use, and suitable for POCT applications.

Carbohydrate-protein template synthesized high mannose loading gold nanoclusters: A powerful fluorescence probe for sensitive Concanavalin A detection and specific breast cancer cell imaging.

Protein-encapsulated gold nanoclusters (Au NCs) have recently gained much attention in biosensing and bioimaging applications owing to their remarkable fluorescence properties, nontoxicity and good biocompatibility. In this work, the mannose was grafted onto the bovine serum albumin (BSA) encapsulated Au NCs (BSA-Au NCs) to produce a mannose functionalized BSA-Au NCs (Man-BSA-Au NCs) as a new fluorescence probe for Concanavalin A (Con A) detection and human breast cancer cell imaging. A new strategy with mannose-BSA conjugates as template was firstly applied for the synthesis of Man-BSA-Au NCs, leading to a high loading of mannose (767.6 ± 7.2 mg/L) onto BSA-Au NCs. The as-prepared Man-BSA-Au NCs showed advantages of facile preparation, good monodispersity and strong red-emission. Notably, aggregation-induced fluorescence quenching of Man-BSA-Au NCs was triggered by Con A due to the multivalent cooperative interactions between mannose and Con A, which was subsequently confirmed by MALDI-TOF MS. Hence highly selective and sensitive fluorescence detection of Con A was achieved by using Man-BSA-Au NCs as a fluorescence sensor. A good linear relationship was obtained over the range of 0.01-1 µM (R2 = 0.994) with a detection limit of 0.62 nM (S/N = 3). The developed sensor was then applied to determine Con A in human serum with acceptable recoveries of 93.70-104.8%. Moreover, based on the specific recognition between mannose and overexpressed mannose receptors on human breast cancer cells, the Man-BSA-Au NCs were successfully utilized for cancer cell imaging with good specificity.

Microwave irradiation-assisted high-efficiency N-glycan release using oriented immobilization of PNGase F on magnetic particles.

Peptide-N-glycosidase F (PNGase F) is the most frequently used enzyme to release N-glycan from glycoproteins in glycomics; however, the releasing process using PNGase F is tedious and can range in duration from hours to overnight. Recently, efforts have been made to accelerate this enzymatic reaction, and they include the use of microwave irradiation, ultrahigh pressure, enzyme immobilization, and other techniques. Here, we developed a novel method combining the oriented immobilization of PNGase F on magnetic particles and microwave-assisted enzymatic digestion techniques to achieve highly efficient release of N-glycans. The oriented immobilization of PNGase F on magnetic particles utilizes the affinity of its co-expressed His-tag towards iminodiacetic acid-Nickel modified magnetic particles. Compared with non-oriented immobilization, the oriented immobilization of PNGase F exhibits several advantages including tolerance to high temperature (52 °C) and the ability to retain strong activity after more than five reuses. When used in combination with microwave irradiation, efficient N-glycan removal from ribonuclease B was achieved within 5 min. The proposed strategy was also used to release glycan from fetuin and human serum and has proven to provide a promising deglycosylation method for the characterization of protein glycosylation.

Development of a filter-aided extraction method coupled with glycosylamine labeling to simplify and enhance high performance liquid chromatography-based N-glycan analysis.

Efficient sample pretreatment of N-glycans from glycoproteins is essential but challenging due to the limitations of existing tedious and laborious methods in N-glycomics. This study aimed to establish a filter-aided extraction method coupled with glycosylamine AQC labeling for a simple and rapid direct HPLC-FLD-based analysis of N-glycans. The developed method was demonstrated to be simpler and more sensitive compared to previous HILIC SPE purification method coupled with glycosylamine labeling. It has been validated with wild-type N-glycans from human transferrin and RNase B and then was successfully applied to investigate N-glycan profiles of the transferrin in human serum and a monoclonal antibody (mAb). Results showed good applicability of the method for complex samples. Additionally, this method is compatible with the replicate determination of N-glycan samples to assess the high-throughput analysis of glycan variability in mAb sample.

Agarose-based microwell array chip for high-throughput screening of functional microorganisms.

Natural bacterial communities provide a rich source of biocatalysts, however, high-throughput screening for the functional bacteria remains a major challenge. Here, we present an agarose-based microwells array chip for functionally screening and isolating novel microorganisms with merits of high-throughput, high specificity and sensitivity. In this approach, the bacterial cells were loaded with single cell per a microwell mode and were incubated in the arrayed agarose microwells. The growths of single cells were then monitored in real time and the enzyme reaction activities were assessed at the individual microwell resolution. To validate the reliability of the method, we subsequently applied it to screen lipase-producing bacteria from the pond water based on lipase hydrolysis of the substrate in the presence of a fluorescent dye (rhodamine B), which emitted yellow-orange fluorescent halos upon UV-light irradiation. A total amount of more than 310,000 isolates from pond water could be screened at a time in only 13 h with reduced consumption of reagents. As a result, 12 microcolonies were identified out with the desired phenotype, in which two new species were discovered based on 16S rRNA sequencing. We expect the developed method to be potentially useful to high-throughput analysis, microbiology and synthetic biology.

Cellulose microspheres-filled pipet tips for purification and enrichment of glycans and glycopeptides.

The pretreatment of low-abundance glycans/glycopeptides prior to mass spectrometry (MS) analysis is of great significance for glycosylation identification of proteins. Herein, a simple one-step purification and enrichment procedure of glycans and glycopeptides was achieved using cellulose microspheres-filled pipet tips (CM-PTs). CM-PTs were fabricated by conveniently filling cellulose microspheres synthesized by sol-gel transition method into 100 µL pipet tips. The whole enrichment and purification process were completed within two minutes by pipetting up and down for dozens of times (30-40), followed by the direct analysis of MALDI TOF-MS. Upon the assessment of model glycoproteins, including human immunoglobulin G (IgG) and horse radish peroxidase (HRP), the CM-PT method exhibited good selectivity (IgG/BSA = 1:100), high sensitivity (1 ng µL-1 of IgG), large binding capacity (125 mg g-1, IgG/cellulose microspheres) and excellent reproducibility in glycopeptide enrichment. The capability for purifying glycans of CM-PTs was also investigated, presenting high binding capacity (8 mg g-1, maltoheptaose/cellulose microspheres) and good recoveries (81.3-89.8%). The developed in-pipet-tip system shows a great potential in efficient and high-throughput analysis of protein glycosylation.

Determination of N-glycans by high performance liquid chromatography using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as the glycosylamine labeling reagent.

Robust, efficient identification and accurate quantification of N-glycans are of great significance in N-glycomics analysis. Here, a simple and rapid derivatization method, based on the combination of microwave-assisted deglycosylation and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) labeling, was developed for the analysis of N-glycan by high performance liquid chromatography with fluorescence detection (HPLC-FLD). After optimizing various parameters affecting deglycosylation and derivatization by RNase B, the time for N-glycan labeling was shortened to 50 min with ∼10-fold enhancement in detection sensitivity comparing to conventional 2-aminobenzoic acid (2-AA) labeling method. Additionally, the method showed good linearity (correlation coefficients > 0.991) and reproducibility (RSD < 8.7%). These advantages of the proposed method were further validated by the analysis of complex samples, including fetuin and human serum. Investigation of serum N-glycome for preliminary diagnosis of human lung cancer was conducted, where significant changes of several N-glycans corresponding to core-fucosylated, mono- and disialylated glycans have been evidenced by a series of statistical analysis.