Portable droplet-based real-time monitoring of pancreatic α-amylase in postoperative patients.

Postoperative complications after pancreatic surgery are frequent and can be life-threatening. Current clinical diagnostic strategies involve time-consuming quantification of α-amylase activity in abdominal drain fluid, which is performed on the first and third postoperative day. The lack of real-time monitoring may delay adjustment of medical treatment upon complications and worsen prognosis for patients. We report a bedside portable droplet-based millifluidic device enabling real-time sensing of drain α-amylase activity for postoperative monitoring of patients undergoing pancreatic surgery. Here, a tiny amount of drain liquid of patient samples is continuously collected and co-encapsulated with a starch reagent in nanoliter-sized droplets to track the fluorescence intensity released upon reaction with α-amylase. Comparing the α-amylase levels of 32 patients, 97 % of the results of the droplet-based millifluidic system matched the clinical data. Our method reduces the α-amylase assay duration to approximately 3 min with the limit of detection 7 nmol/s·L, enabling amylase activity monitoring at the bedside in clinical real-time. The presented droplet-based platform can be extended for analysis of different body fluids, diseases, and towards a broader range of biomarkers, including lipase, bilirubin, lactate, inflammation, or liquid biopsy markers, paving the way towards new standards in postoperative patient monitoring.

High-Throughput Gel Microbeads as Incubators for Bacterial Competition Study.

Bacteria primarily live in structured environments, such as colonies and biofilms, attached to surfaces or growing within soft tissues. They are engaged in local competitive and cooperative interactions impacting our health and well-being, for example, by affecting population-level drug resistance. Our knowledge of bacterial competition and cooperation within soft matrices is incomplete, partly because we lack high-throughput tools to quantitatively study their interactions. Here, we introduce a method to generate a large amount of agarose microbeads that mimic the natural culture conditions experienced by bacteria to co-encapsulate two strains of fluorescence-labeled Escherichia coli. Focusing specifically on low bacterial inoculum (1-100 cells/capsule), we demonstrate a study on the formation of colonies of both strains within these 3D scaffolds and follow their growth kinetics and interaction using fluorescence microscopy in highly replicated experiments. We confirmed that the average final colony size is inversely proportional to the inoculum size in this semi-solid environment as a result of limited available resources. Furthermore, the colony shape and fluorescence intensity per colony are distinctly different in monoculture and co-culture. The experimental observations in mono- and co-culture are compared with predictions from a simple growth model. We suggest that our high throughput and small footprint microbead system is an excellent platform for future investigation of competitive and cooperative interactions in bacterial communities under diverse conditions, including antibiotics stress.

Population dynamics of cross-protection against ß-lactam antibiotics in droplet microreactors.

Introduction: Bacterial strains that are resistant to antibiotics may protect not only themselves, but also sensitive bacteria nearby if resistance involves antibiotic degradation. Such cross-protection poses a challenge to effective antibiotic therapy by enhancing the long-term survival of bacterial infections, however, the current understanding is limited. Methods: In this study, we utilize an automated nanoliter droplet analyzer to study the interactions between Escherichia coli strains expressing a ß-lactamase (resistant) and those not expressing it (sensitive) when exposed to the ß-lactam antibiotic cefotaxime (CTX), with the aim to define criteria contributing to cross-protection. Results: We observed a cross-protection window of CTX concentrations for the sensitive strain, extending up to approximately 100 times its minimal inhibitory concentration (MIC). Through both microscopy and enzyme activity analyses, we demonstrate that bacterial filaments, triggered by antibiotic stress, contribute to cross-protection. Discussion: The antibiotic concentration window for cross-protection depends on the difference in ß-lactamase activity between co-cultured strains: larger differences shift the 'cross-protection window' toward higher CTX concentrations. Our findings highlight the dependence of opportunities for cross-protection on the relative resistance levels of the strains involved and suggest a possible specific role for filamentation.

Coexistence of fluorescent Escherichia coli strains in millifluidic droplet reactors.

Understanding competition and cooperation within microbiota is of high fundamental and clinical importance, helping to comprehend species' evolution and biodiversity. We co-encapsulated and cultured two isogenic Escherichia coli strains expressing blue (BFP) and yellow (YFP) fluorescent proteins into numerous emulsion droplets and quantified their growth by employing fluorescence measurements. To characterize and compare the bacterial growth kinetics and behavior in mono and co-culture, we compared the experimental observations with predictions from a simple growth model. Varying the initial ratio (R0) of both cell types injected, we observed a broad landscape from competition to cooperation between both strains in their confined microenvironments depending on start frequency: from a nearly symmetric situation at R0 = 1, up to the domination of one subpopulation when R0 ≫ 1 (or R0 ≪ 1). Due to competition between the strains, their doubling times and final biomass ratios (R1) continuously deviate from the monoculture behavior. The correlation map of the two strains' doubling times reveals that the R0 is one of the critical parameters affecting the competitive interaction between isogenic bacterial strains. Thanks to this strategy, different species of bacteria can be monitored simultaneously in real-time. Further advantages include high statistical output, unaffected bacteria growth, and long-time measurements in a well-mixed environment. We expect that the millifluidic droplet-based reactor can be utilized for practical clinical applications, such as bacterial antibiotic resistance and enzyme reaction kinetics studies.

Electrospinning design of functional nanostructures for biosensor applications.

Electrospinning represents a simple and effective strategy for fabricating nanofibrous structures and materials with large surface-to-volume ratios and desirable engineered properties. Thus, incorporating nanoscale building blocks (NBBs) like nanoparticles, graphene quantum dots, carbon nanotubes, and graphene into electrospun fibers has become one of the most attention-getting research topics in the field of biosensing. However, the dispersion behavior of NBBs in the nanofibers, the limited surface area of nanofibers and the insufficient immobilization sites for tested biomolecules still restrict the better performances and broader applications of the fabricated biosensors. In this review, we present a comprehensive survey of strategies that have been utilized to fabricate functional fibrous nanostructures for the amplification of the detection signals of nanostructure-based biosensors. In particular, from the perspective of design configuration, we systematically summarized recent advances in the electrospinning fabrication of hybrid polymer nanofibers decorated with functionalized NBBs. The strategies for promoting better dispersion of NBBs in electrospun nanofibers, including direct blending before electrospinning and in situ synthesis during electrospinning, are introduced in detail. In addition, some effective processing routes for increasing immobilization sites of tested biomolecules such as arrangement of NBBs and morphological processing of nanofibers are also presented. In addition, the suitability of electrospun nanostructures for biosensors, and the advantages and disadvantages of each method for improving the biosensing performance are also discussed.

Recent advances in the fabrication and structure-specific applications of graphene-based inorganic hybrid membranes.

The preparation and applications of graphene (G)-based materials are attracting increasing interests due to their unique electronic, optical, magnetic, thermal, and mechanical properties. Compared to G-based hybrid and composite materials, G-based inorganic hybrid membrane (GIHM) offers enormous advantages ascribed to their facile synthesis, planar two-dimensional multilayer structure, high specific surface area, and mechanical stability, as well as their unique optical and mechanical properties. In this review, we report the recent advances in the technical fabrication and structure-specific applications of GIHMs with desirable thickness and compositions. In addition, the advantages and disadvantages of the methods utilized for creating GIHMs are discussed in detail. Finally, the potential applications and key challenges of GIHMs for future technical applications are mentioned.

Electrospinning graphene quantum dots into a nanofibrous membrane for dual-purpose fluorescent and electrochemical biosensors.

Graphene quantum dots (GQDs) have become increasingly important for applications in energy materials, optical devices and biosensors. Here we report a facile technique to fabricate a nanofibrous membrane of GQDs by electrospinning water-soluble GQDs with polyvinyl alcohol (PVA) directly. The structure and fluorescence properties of the fabricated PVA/GQD nanofibrous membrane were investigated using scanning and transmission electron microscopy, and fluorescence microscopy. It was found that the electrospun PVA/GQD nanofibrous membrane has a three-dimensional structure with a high surface area to volume ratio, which is beneficial for the adsorption of electrolytes and the diffusion of reactants. For the first time, the created PVA/GQD nanofibrous membrane was utilized to fabricate dual-purpose fluorescent and electrochemical biosensors for highly sensitive determination of hydrogen peroxide (H2O2) and glucose. The experimental results indicated that the fluorescence intensity of the nanofibrous membrane decreased linearly with increasing H2O2 concentration, because the addition of H2O2 leads to fluorescence quenching of the GQDs, which endows the fabricated nanofibrous membrane with fluorescence activity. Besides, after binding glucose oxidase onto the created nanofibrous membrane, the fabricated nanofibrous membrane showed high sensitivity and selectivity for glucose detection. In addition, the PVA/GQD nanofibrous membrane can also be directly electrospun onto an electrode for electrochemical detection of H2O2. This novel nanofibrous membrane exhibits excellent catalytic performance and fluorescence activity, and therefore has potential applications for the highly stable, sensitive, and selective detection of H2O2 and glucose.

Electrospun doping of carbon nanotubes and platinum nanoparticles into the ß-phase polyvinylidene difluoride nanofibrous membrane for biosensor and catalysis applications.

A novel ß-phase polyvinylidene difluoride (PVDF) nanofibrous membrane decorated with multiwalled carbon nanotubes (MWCNTs) and platinum nanoparticles (PtNPs) was fabricated by an improved electrospinning technique. The morphology of the fabricated PVDF-MWCNT-PtNP nanofibrous membrane was observed by scanning electron microscopy, and the formation of high ß-phase in the hybrid nanofibrous membrane was investigated by Fourier transform infrared spectroscopy and differential scanning calorimetry. The uniform dispersion of MWCNTs and PtNPs in the PVDF hybrid nanofibrous membrane and their interaction were explored by transmission electron microscopy and X-ray diffraction. For the first time, we utilized this created PVDF-MWCNT-PtNP nanofibrous membrane for biosensor and catalysis applications. The nonenzymatic amperometric biosensor with highly stable and sensitive, and selective detection of both H2O2 and glucose was successfully fabricated based on the electrospun PVDF-MWCNT-PtNP nanofibrous membrane. In addition, the catalysis of the hybrid nanofibrous membrane for oxygen reduction reaction was tested, and a good catalysis performance was found. We anticipate that the strategies utilized in this work will not only guide the further design of functional nanofiber-based biomaterials and biodevices but also extend the potential applications in energy storage, cytology, and tissue engineering.