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A multivalent DNA walker amplification biosensor combined with the microfluidic chip for simultaneously and sensitively detecting V.P, S.T, and S.A bacteria.

An exquisitely designed biomimetic multienzyme hybrid has been constructed by in situ encapsulating enzymes within a hierarchically porous bimetallic metal–organic framework.

Fc-MBL@Fe 3 O 4 capture not only live bacteria in aqueous humor but also bacteria inactivated by antibiotics. Fc-MBL@Fe 3 O 4 combined with micro-LB broth culture significantly reduced the turnaround time (TAT) by more than half a day by shortening the time required for bacterial identification.

When nanoparticles (NPs) come into contact with bioenvironments, a protein corona forms on the NP surface. Previous reports showed that the constituents of the corona change with time. However, how different protein corona compositions influence cells, especially immune cells, has received less attention. Macrophages are important immune cells that can be polarized into a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. In this study, AuNPs were incubated with human plasma for different periods to obtain time-related AuNP-coronas, and the influences of time-related AuNP-coronas on macrophage polarization were investigated. The macrophage morphology, biomarkers, cytokine secretion studies show that the pristine AuNPs and 4 h-AuNP-corona induced macrophage cells into M2 phenotype, while the co-incubation of 12 h-AuNP-corona and macrophage cells result in M1 phenotype. Further proteomic analysis showed that the compositions of protein corona were changing constantly after AuNPs contacted with plasma. When the incubation time increased to 12 h, the immune proteins in protein corona were increased significantly, which play a key role in modulation of the different macrophages polarization. Our findings demonstrated that plasma incubation time is an important parameter that needs to be taken into account in the study of nano-immune interactions and safe use of NPs in biological systems. Moreover, our finding can be a new efficient strategy for activating inflammatory or anti-inflammatory in medical treatment.

Live foodborne pathogens proliferate rapidly and do great harm to human health, which requires appropriate methods to supervise. In this work, a portable adenosine triphosphate (ATP) bioluminescence sensor with high specificity for live E. coli O157:H7 strain synergistically enhanced by orientated phage-modified stir bar extraction and bio-proliferation was developed. In brief, the selected phages were directionally immobilized on the poly(diallyldimethylammonium chloride)-modified gold stir bar as the bioreceptor. Following the simple stir bar absorptive extraction and bio-proliferation in the Luria-Bertani medium, the number of captured E. coli O157:H7 exploded. Finally, it was quantified by a portable ATP bioluminescence sensor. Benefitting from the high specificity of phage and simple signal dual-amplification strategy, the proposed biosensor achieved the recognition of live bacteria at strain level with superior sensitivity. Also, the portable signal readout made it suitable for on-site detection. Under optimal conditions, this bioassay provided a detectable range of 10

Macrophage cells are plastic and can be polarized into opposing phenotypes, pro-inflammatory (M1-like cells) or anti-inflammatory (M2-like cells). Reprograming of M2-like cells into M1 phenotype wi...

The analysis of positional isomers is of great significance because their different chemical properties but similar structures make separation difficult. In this work, a simple method for simultaneously discriminating three positional isomers of 2-aminobiphenyl (2-ABP), 3-ABP, and 4-ABP was studied by ion mobility spectrometry (IMS) and quantum mechanical calculations at the molecular level. In the experiments, three ABP isomers were mixed with α-, β-, and γ-cyclodextrins (CD), and the IMS results show that the three ABP isomers were clearly recognized by the formed complex of [α-CD + ABP + H]+ via measuring their IMS, in which the different ion mobilities of 1.515, 1.544, 1.585 V·s·com-2 with the collision cross sections (CCS) of 307.3, 312.5, 320.8 A2 were obtained for [α-CD + 2-ABP + H]+, [α-CD + 3-ABP + H]+, and [α-CD + 4-ABP + H]+, respectively. Collision induced dissociation analysis of the three [α-CD + ABP + H]+ isomer complexes were further studied, indicating that the same fragmentation process required different collisional energies, and the greater the CCS for the [α-CD + ABP + H]+ with looser structure and the smaller energy required. Besides, the favorable conformation and the CCS value of the different [CD + ABP + H]+ isomer complexes were measured via quantum mechanical calculations to detail their intermolecular interactions. It revealed that the intermolecular binding between 2-ABP and α-CD is different from that of 3- and 4-ABP, resulting in different molecular conformations and CCS, and the interaction modes of ABP with β-CD are similar to that with γ-CD, which are very consistent with the experimental observations. Finally, relative quantification of the method was performed, and satisfactory linearity with correlation coefficients (R2) greater than 0.99 was obtained. This method for isomer discrimination and conformation analysis possesses the advantages of simplicity, sensitivity, cost-effectiveness, and as such it may be widely applied in chemistry and pharmaceutical sciences.

The direct analysis of glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) presents limited sensitivity due to the lower ionization efficiency of glycans. Various chemical derivatization methods have been developed to improve the detection sensitivity of glycans, but most of them need tedious preparation and cleanup procedures. Herein, a reactive matrix, 4-hydrazinoquinazoline (4-HQ), was developed for the rapid and sensitive detection of both neutral and sialylated glycans by MALDI MS. With 4-HQ as the reactive matrix, the detection limits of maltoheptaose and A3 glycan decreased 100-fold and 20-fold, respectively, compared with the conventional matrix. Moreover, 4-HQ formed homogeneous crystals and therefore showed good shot-to-shot reproducibility. Finally, the reactive matrix was successfully applied for the analysis of glycans released from glycoproteins and human serum. Importantly, the application of 4-HQ is the same as that of a conventional matrix with the additional advantage of on-target reaction at room temperature. Thus, 4-HQ can be used for the routine analysis of glycans by MALDI MS due to its simple use, great reproducibility, and enhanced detection of both neutral and sialylated glycans.

The combination of bio- and chemo-catalysts for sequential cascades has received considerable attention in analytical fields because of the regulable catalytic efficiency and selectivity under various physiological conditions. In this paper, a versatile multienzyme cascade nanoplatform with excellent activity for biosensing is demonstrated by combining metal-organic framework (MOF)-based nanozyme with natural enzymes. A boronic acid-modified MOF, MIL-100(Fe)-BA, was obtained

MALDI-TOF MS is well-recognized for microbial identification and widely used in research and clinical fields due to its specificity, speed of analysis, and low cost of consumables. However, the classification or identification accuracy is poor for E. coli and Shigella. In addition, FTIR is a promising tool for bacterial typing. In this study, 14 strains of E. coli and 9 strains of Shigella were typed by both MALDI-TOF MS and FTIR techniques. Alternatively, a data fusion strategy using these two approaches was attempted to achieve better typing accuracy. Hierarchical clustering analysis (HCA) revealed that the typing accuracies for selected E. coli and Shigella from blood agar were 65.2%, 78.3%, and 100% for MALDI-TOF MS, FTIR, and FTIR combined with MALDI-TOF MS, respectively. Based on these results, a library constructed of the MS-IR fusion data was employed to identify individual bacteria at the strain level, and the library-based typing accuracies for 207 test spectra of sample strains from three different culture media yielded 97.6% accuracy for distinguishing between E. coli and Shigella at genus level. For species level and strain level, it yielded 95.2% and 92.3% typing accuracy, respectively. These results indicate that FTIR is a better technique for typing selected E. coli and Shigella than MALDI-TOF MS, and the accuracy is increased by combining the data from MALDI-TOF MS and FTIR spectroscopy. FTIR could be used to supplement MALDI-TOF MS for the identification and typing of taxonomic microorganisms.

Carbon dots (CDs) have attracted tremendous attention for their outstanding advantages in luminescence. Here, α-amino-substituted lysine derivatives with the determined chemical structure were employed as precursors to obtain bright and highly stable fluorescent CDs through a facile hydrothermal route. The relationships among the chemical structure of precursors, CD fluorescence, and particle size were investigated. The results indicated that increased numbers of functional groups in precursors could promote the degree of cross-linking and lead to a smaller size, better fluorescent properties, and stronger stability of CDs. The C-CDs that were prepared from lysine derivatives with most functional groups showed excitation-dependent dual excitation and dual emission (DE2), high-stability luminescence, strong resistance to photobleaching, and high selectivity to Fe3+ and could be used as a sensitive probe for Fe3+ detection.

Tumor-derived exosomes, which contain RNA, DNA, and proteins, are a potentially rich non-invasive source of biomarkers. However, no efficient isolation or detection methods are yet available. Here, we developed a microfluidic Raman biochip designed to isolate and analyze exosomes in situ. Anti-CD63 magnetic nanoparticles were used to enrich exosomes through mixing channels of a staggered triangular pillar array. EpCAM-functionalized Raman-active polymeric nanomaterials (Raman beads) allow rapid analysis of exosome samples within 1 h, with a quantitative signal at 2230 cm-1. The limit of detection of this biochip approaches 1.6 × 102 particles per mL with 20 μL samples. The newly developed biochip assay was successfully applied in the determination of exosomes from clinical serum samples. Thus, this novel device may have potential as a clinical exosome analysis tool for prostate cancer.

Conventional microfluidics with a solid mixer for exosome detection is constrained by the low binding efficiency of the solid-liquid boundary effects and reduced sensitivity of individual markers. Here, we report a 3D-SiO

Macrophage cells are plastic and can be polarized into opposing phenotypes, pro-inflammatory (M1-like cells) or anti-inflammatory (M2-like cells). Reprograming of M2-like cells into M1 phenotype will contribute significantly to combatting cancer. Gold nanoparticles (AuNPs) are intensively studied in various fields for their distinctive photo-chemical properties. However, the immune response of AuNPs is still unclear. In this study, AuNPs and CaCO

Bacterial FT-IR signals are extremely specific and highly reproducible, making FT-IR an efficient tool for bacterial typing at the subspecies level. The polysaccharide and nucleic acid FT-IR regions (1200-900 cm

Functional bacterial enrichment magnetic beads (Fe

Differentiating methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA and MSSA) is crucial for clinical diagnosis and anti-microbial treatment. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is an efficient tool for identifying pathogenic microorganisms at the bacterial species level. Here, we found that MRSA and MSSA can be differentiated by MALDI-TOF MS by employing (E)-propylα-cyano-4-hydroxyl cinnamylate (CHCA-C3) as the matrix, which shows great performance for proteins/peptides, especially hydrophobic proteins. The results show that the mass spectra profile of standard MRSA (ATCC 43300) is significantly different from the profiles of standard MSSA strains (ATCC 25923 and 29213) when using CHCA-C3 as the matrix compared to traditional matrix. The mass profiles had great reproducibility and were scarcely influenced by the growth medium. Due to the enhanced discrimination ability of CHCA-C3, we collected the mass spectra of 62 clinical S. aureus strains and selected four representative peaks for principal component analysis, which showed great differentiation. Our results suggest that employing a suitable matrix could enhance the discrimination ability of antibiotic-resistant bacteria by MALDI-TOF MS.

Infrared (IR) spectroscopy is a highly sensitive technique that provides complete information on chemical compositions. The IR spectra of proteins or peptides give rise to nine characteristic IR absorption bands. The amide I bands are the most prominent and sensitive vibrational bands and widely used to predict protein secondary structures. The interference of H

Accurate and effective discrimination of E. coli and Shigella is an important clinical issue, and there are many limitations in traditional methods of analysis. FT-IR shows great potential in the classification of bacteria with high specificity and low cost. In this study, we evaluated the efficiency of this technique when combined with multivariate analysis for rapid classification of E. coli and Shigella, which is difficult using traditional analytical methods. Machine learning and statistical tools were employed in combination with FT-IR to classify 14 E. coli and 9 Shigella strains. The classification accuracies for select E. coli and Shigella strains from blood agar were 0.7826, 0.8696, and 0.9565 at the genus, species, and strain levels, respectively. In addition, we used the FT-IR data of select strains from three different culture media for cross-validation, yielding an accuracy of 0.3681 at the strain level. These results indicate that the bacterial culture conditions have a significant impact on the FT-IR patterns. Based on this, an improved strategy for training an ensemble classifier model considering bacterial culture factors was constructed, resulting in almost perfect separation with an accuracy of 0.9394 for strain-level classification. These results show the potential of FT-IR combined with multivariate analysis for more reliable bacterial classification.

Tumor exosomes that inherit specific molecules from their parent cells are emerging as ideal biomarkers in cancer diagnostics. Most currently available exosome isolation and detection methods are time-consuming and non-specific; thus, rapid and specific exosome detection methods are needed both clinically and in research. Here, a dual-functional platform is reported composed of reversible conjunction and “off-on” signal responses. Fe3O4@SiO2@TiO2 particles with high affinity were applied to capture exosomes, and model exosomes could be isolated from solution within 20 min with a capture efficiency of 91.5%. An “on-off” fluorescence response PSMA aptasensor was constructed with improved selectivity to detect tumor exosomes by recording the fluorescence intensity with λex/em = 557/580 nm. The standard curve for detecting tumor exosomes with the aptasensor was calculated as y = 371.7x + 66.17, ranging from 0.05 to 1 × 104 particles/μL, with R2 = 0.9737, and a detection limit of 5 × 102 particles/μL in solution. This method was successfully applied to clinical samples, and the results showed better performance in distinguishing prostate cancer patients and healthy samples than the traditional nanoparticle-tracking analysis (NTA) method. This rapid and accurate detection method for prostate cancer may aid in rapid clinical diagnosis.

Linaridins are a small but growing class of natural products belonging to the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. The class A linaridins, exemplified by cypemycin, possess an unusual S-[( Z)-2-aminovinyl]-d-cysteine (AviCys) residue. Formation of the AviCys in cypemycin requires an oxidative decarboxylation of the precursor peptide C-terminal Cys, and this reaction is catalyzed by a flavin-dependent decarboxylase CypD. In this work, we investigate the molecular recognition processes of CypD by a combination of computational and biochemical analysis. We show that the substrate binding clamp of CypD undergoes dramatic fluctuation, mediating both the substrate entrance into and product release from the catalytic pocket. Extensive molecular dynamic simulations and Fourier transform IR analyses indicated that binding of the substrate induces substantial structural change of the enzyme, converting the substrate-binding clamp from a random loop to a more ordered structure comprising two β sheets and a β turn. The salt bridge between Arg159 guanine and the Cys carboxylate of substrate plays an important role in mediating substrate binding, while hydrophobic interactions are also important in this process. These results provide important mechanistic insights into CypD and other flavin-dependent Cys decarboxylases, and could facilitate future biosynthetic and bioengineering efforts in studying AviCys-containing RiPPs.

S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) is a unique motif found in several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs). Biosynthesis of AviCys requires flavin-dependent Cys decarboxylases, which are highly divergent among different RiPP classes. In this study, we solved the crystal structure of the cypemycin decarboxylase CypD. We show that CypD is structurally highly similar to lanthipeptide decarboxylases despite the absence of sequence similarities between them. We further show that Cys decarboxylases from four RiPP classes have evolved independently and form two major clusters. These results reveal the convergent evolution of AviCys biosynthesis and suggest that all the flavin-dependent Cys decarboxylases likely have a similar Rossmann fold despite their sequence divergences.

Cholesterol is a vital building block for animal cell membranes and participates in the synthesis of various hormones. Accurate quantitation of cholesterol in food is crucial for healthy diets. Here, we describe an enzyme-assisted reactive matrix-assisted laser desorption/ionization mass spectrometry (ReMALDI-MS) assay for the quantification of cholesterol in food. First, cholesterol was converted to 4-cholesten-3-one using the cholesterol oxidase, and then reacted with a reactive matrix, 4-hydrazinoquinazoline (4-HQ), to form a hydrazone bond. Utilizing 4-HQ significantly improved the ionization efficiency of cholesterol, which possesses poor ionization efficiency in MALDI-MS, and no additional tedious derivatization/purification steps were needed. Thus, the proposed assay was successfully used for the quantification of cholesterol in bovine milk and cream. The standard recovery tests show a recovery range of 95.3-103.0% with a relative standard deviation of 0.3-3.1%. Therefore, the proposed enzyme-assisted ReMALDI-MS assay has great potential for quantification of cholesterol in other foods.

Chiral recognition is of highly interest in the areas of chemistry, pharmaceuticals, and bioscience. An effective strategy of enantiomeric determination of amino acids (AAs) was developed in this work. All 19 natural AAs enantiomers can be easily distinguished by ion mobility-mass spectrometry of the non-covalent complexes of AAs with cyclodextrins (α-CD, β-CD and γ-CD) and Mg

Enantiomeric drugs are widely used and play important roles in pharmaceuticals. Ion mobility spectrometry coupled with MS technology provides a unique method for distinguishing the enantiomeric drugs, enantiomeric identification and quantitation in the gas phase. In this study, enantiomeric molecules of ibuprofen and flurbiprofen were clearly recognized by forming host-guest complex ions using trapped ion mobility time-of-flight MS. Ternary complex ions can be produced easily by electrospray ionization of the mixed solutions of ibuprofen, cyclodextrins, and CaCl2 , LiCl, or NaCl, as well as flurbiprofen, cyclodextrins, and CaCl2 . The relative contents of different chiral ibuprofens in a mixed solution were also quantitatively measured. This new method is a simple, effective, and a convenient enantioselective analysis method. This article is protected by copyright. All rights reserved.

Rationale The rapid identification of small-molecule chiral drugs is challenging due to subtle structural differences. Different enantiomers of chiral drugs may produce inverse biological effects through their different pharmacokinetics. Therefore, it is highly desirable to distinguish the chirality of drug molecules. METHODS The chirality of pregabalin was distinguished by studying the ion mobility spectra of the ternary non-covalent complexes formed with cyclodextrins (CDs), pregabalin, and alkali-earth cations using trapped ion mobility spectrometry (TIMS). The ternary non-covalent complex ions were determined by electrospray ionization of mixed solutions. The analyzed sample was simply mixed, without derivatization or sample pretreatment. The relative contents of pregabalin enantiomers were derived using a calibration curve method. RESULTS The ion mobility spectra of several ternary non-covalent complexes formed with α-, β-, and γ-CD, pregabalin, and alkali-earth cations were obtained. We compared their ability to distinguish the chirality of pregabalin. The best peak-to-peak resolution (Rp-p ) was estimated to be 2.20 for [2β-CD + pregabalin + Sr]2+ , which can be ascribed as baseline separation. The derived relative contents for S-pregabalin were in agreement with the actual contents. CONCLUSIONS A novel and convenient method for discriminating the chirality of the pregabalin molecule by TIMS was developed and optimized. The chirality of pregabalin was recognized by studying the ion mobility spectra of the ternary non-covalent complexes, such as [2β-CD + pregabalin + Sr]2+ . This TIMS method could also be used to quantify the relative contents of pregabalin enantiomers.

Direct identification of bacteria in blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is interfered with by a variety of non-bacterial proteins derived from blood cells and culture media. Thus, appropriate pre-treatments are needed for successful identification. Here, the bacteria in blood culture bottles were enriched using co-magnetic beads and processed for MALDI-TOF MS profiling. In this strategy, the Fc-containing mannose-binding lectin-coated Fe3O4 (Fc-MBL@Fe3O4) is incorporated with human IgG-coated Fe3O4 (IgG@Fe3O4) to form co-magnetic beads, which can recognize both Gram-positive and Gram-negative bacteria. Compared to single magnetic beads Fc-MBL@Fe3O4 or IgG@Fe3O4, co-magnetic beads resulted in better bacterial capture efficiency and, therefore, could decrease the false-negative results. Our proposed strategy is much more suitable for enrichment of clinically unknown bacteria from blood culture bottles for MALDI-TOF MS database identification.

Positional isomer recognition is a challenging scientific issue. Fast and accurate detection of isomers is required for understanding their chemical properties. Here, we describe a method for simultaneous recognition of three positional isomers of 2-aminobenzenesulfonic acid (2-ABSA), 3-ABSA, and 4-ABSA using trapped ion mobility spectroscopy-time-of-flight mass spectrometry (TIMS-TOF-MS). The three ABSA positional isomers were recognized by measuring the different ion mobility of the ternary complexes of [β-cyclodextrin (CD)+ABSA + Li]+ or [λ-CD + ABSA + Na]+, because their different collision cross-sections or different spatial conformations. The collision-induced dissociation mechanism of the different complexes of [β-CD + ABSA + Li]+ and [λ-CD + ABSA + Na]+ using tandem mass spectrometry exhibited the same dissociation process with slightly different dissociation energies, which the smaller cross-section requires higher collision energy that means the smaller complex with tighter and more stable conformation than a larger complex for the ABSA complexes. In addition, relative quantification of the ABSA isomers was studied by measuring any two of the three ABSA isomer complexes at different molar ratio of 10:1 to 1:10 in the μM range, good linearity (R2 > 0.99) with precision between 2.14% and 2.58%, and accuracy ≥ 97.1% were obtained. The method for fast determination and recognition of ABSA positional isomers by combination with CD and alkali metal ions possesses the advantages of being simple, direct, rapid, sensitive, cost-effective, and needs no chemical derivatives or chromatographic separation before analysis. Therefore, the proposed method would be a powerful tool for the analysis of ABSA isomers or even other positional isomers.

Bacterial infections are the key cause of morbidity and mortality worldwide. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS)-based bacterial identification has been widely accepted in the clinic. Functional material, such as rabbit immunoglobulin G-modified Fe3O4 (IgG@Fe3O4) and fragment crystallizable mannose binding lectin-modified Fe3O4 (FcMBL@Fe3O4), is used to capture bacteria from biological samples for MALDI-TOF MS identification, and the bacteria MS signals are usually obtained by directly smearing enriched bacteria on a MALDI target with MALDI matrix solution. However, the accuracy of identification based on MALDI-TOF MS may be affected by the presence of functional molecules, especially proteins, resulting in errors in the comparison with the standard bacterial spectra in the database. Moreover, the long-term presence of the magnetic beads on the MALDI-TOF target may reduce the instrument service life. In this study, we constructed FcMBL@Fe3O4 and used it to capture bacteria from both aqueous solution and bovine blood, and the bacterial identification accuracy based on different target preparation methods was compared. In the presence of Ca2+, the similarity scores for bacteria identified with FcMBL@Fe3O4 were ~88% and ~82% for Staphylococcus. aureus and Escherichia coli, respectively. In the presence of ethylenediaminetetraacetic acid (EDTA), bacteria separate from FcMBL@Fe3O4, resulting in similarity scores of ~96% and ~92% for S. aureus and E. coli, respectively. These results indicate that the functional proteins on the surface of nanoparticles affect the accuracy of identification accuracy based on the MALDI-TOF MS database. Thus, the release of bacteria from the functional material could increase the identification accuracy and be beneficial for maintaining the instrument.

Inorganic nanoparticle-based T

One of the main diseases contributing to human death are malignant tumors. Phototherapy is a promising approach for cancer therapy, and functional nanoparticles with targeting ligands are commonly used to improve the therapeutic efficiency. However, recent studies have shown that nanoparticles in contact with a biological fluid can rapidly form a "protein corona" on their surface, which will remarkably decrease the targeting ability. Here, we describe the preparation of hybrid nanomaterials with Bi