Future of biological aerosol detection

Biological aerosol detection in real time is an urgent civilian and military requirement. Such detection capability will be useful in environmental monitoring, for example, in gathering information in perceived hazardous areas such as housing developments downwind of sewage treatment plants. To be truly functional, the instrument has to operate continuously, 24 h a day and 7 days a week with minimal maintenance and few false alarms. A novel concept is proposed. The system employs a rapid front-end warning/alarming mechanism based on optical technologies that provides useful information for protection decision makers. This is connected to a sample collector that feeds a slower back-end liquid chemistry system that provides analytical results to the medical personnel to assist in prophylaxis and therapy decisions. Experience gained from measuring fluorescence signals of single bacterial spores under flow cytometry (FCM) using UV excitation at 340-360 nm, was applied to concept testing of a prototype instrument, built to do the same for aerosols. This machine was capable of resolving particle size as well as fluorescence intensity of each particle under laboratory and field conditions; it was called the fluorescent aerodynamic particle sizer (FLAPS). This paper describes practical aspects of measuring biological aerosols when the results must be compared to reference samplers that provide culturable or “live” data. Treatment of particle size and fluorescence information is discussed with respect to FLAPS and reference data fidelity. Along with an objective method to evaluate FLAPS data correlation to reference data, an approach for determining limit of detection in the field is discussed. In addressing the back-end detector chemistry, we have prioritized a number of important biological characteristics that must be given to a clinician to help in prophylaxis and therapy decisions. A series of biochemical measurements are proposed to define the threat of a sample and different solutions are given to implement these tests. We predict that the future for biological detection looks promising for fluorescence in situ hybridization (FISH) techniques in identifying microorganisms. A conceptual instrument based on merging FCM and microchip-based analysis is described.     

Cytometric methods for measuring bacteria in water: advantages, pitfalls and applications

Rapid detection of microbial cells is a challenge in microbiology, particularly when complex indigenous communities or subpopulations varying in viability, activity and physiological state are investigated. Flow cytometry (FCM) has developed during the last 30 years into a multidisciplinary technique for analysing bacteria. When used correctly, FCM can provide a broad range of information at the single-cell level, including (but not limited to) total counts, size measurements, nucleic acid content, cell viability and activity, and detection of specific bacterial groups or species. The main advantage of FCM is that it is fast and easy to perform. It is a robust technique, which is adaptable to different types of samples and methods, and has much potential for automation. Hence, numerous FCM applications have emerged in industrial biotechnology, food and pharmaceutical quality control, routine monitoring of drinking water and wastewater systems, and microbial ecological research in soils and natural aquatic habitats. This review focuses on the information that can be gained from the analysis of bacteria in water, highlighting some of the main advantages, pitfalls and applications.     

Immunomagnetic separation using carbonyl iron powder and flow cytometry for rapid detection of <Emphasis Type="Italic">Flavobacterium psychrophilum</Emphasis>

Bacterial cold water disease, caused by Flavobacterium psychrophilum, is a serious problem in the aquaculture industry worldwide. Several methods to prevent and treat cold water disease have been studied. Although detection at the early stage of F. psychrophilum infection is very important for the prevention and treatment of cold water disease, an effective detection method has not yet been developed. The use of flow cytometry (FCM) for the rapid determination of bacterial cell numbers with high sensitivity is beginning to attract attention. Immunomagnetic separation (IMS) has also been used to detect F. psychrophilum. The purpose of the present study was to develop a method to quickly determine the number of bacterial cells by combining the FCM and IMS methods. Because samples can be more effectively concentrated using smaller magnetic beads and stronger magnetism, we used carbonyl iron powder as the magnetic beads for the IMS. The detection level of F. psychrophilum using FCM combined with IMS was 5 orders lower than that using FCM without IMS. The values determined using FCM combined with IMS strongly correlated with those obtained using the colony-counting method, in the range of approximately 10–10⁸ colony-forming units per milliliter. One FCM assay could be completed within 60 s and the total assay time, including sample preparation, was less than 2 h. The combined method of FCM with IMS developed in this study can be used reliably for the rapid detection of F. psychrophilum.     

Analysis of subcellular sized particles. Capillary electrophoresis with post-column laser-induced fluorescence detection versus flow cytometry

Flow cytometry (FCM) and more recently capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) have both been used for subcellular particle analysis but their analytical performance has not been compared. In this work, we compare a commercial FCM with an in-house built CE-LIF instrument using fluorescently labeled microspheres and isolated mitochondria. As evidenced by the relative standard deviation (RSD) of the individual fluorescence intensities, FCM is two-fold better than CE-LIF for microspheres with > or =1.5 x 10(6) molecules of equivalent soluble fluorescein (MESF). However, FCM has a comparatively low signal-to-noise ratio (S/N) and high RSD for microspheres with <1.5 x 10(6) MESF. CE-LIF, on the other hand, produces S/N ratios that are >25 times higher than FCM for all the microspheres tested and a lower RSD for microspheres with <1.5 x 10(6) MESF. When 10-N-nonyl acridine orange (NAO)-labeled mitochondria are analyzed, the S/N ratios of both techniques are similar. This appears to result from photobleaching of NAO-labeled mitochondria as they are detected by the LIF detector of the CE-LIF instrument. Both techniques have a niche in subcellular analysis; FCM has the advantage of collecting data for thousands of particles quickly, whereas CE-LIF consumes less than a nanoliter of sample and provides the electrophoretic mobility for individual particles.     

Rapid and ultrasensitive E. coli O157:H7 quantitation by combination of ligandmagnetic nanoparticles enrichment with fluorescent nanoparticles based two-color flow cytometry

A novel, fast and sensitive determination strategy for E. coli O157:H7 has been developed by combination of ligandmagnetic nanoparticles (LMNPs) enrichment with a fluorescent silica nanoparticles (FSiNPs) based two-color flow cytometry assay (LMNPs@FSiNPs-FCM). E. coli O157:H7 was first captured and enriched through the lectin concanavalin A (Con A) favored strong adhesion of E. coli O157:H7 to the mannose-conjugated magnetic nanoparticles. The enriched E. coli O157:H7 was further specially labeled with goat anti-E. coli O157:H7 antibody modified RuBpy-doped FSiNPs, and then stained with a nucleic acid dye SYBR Green I (SYBR-I). After dual-labeling with FSiNPs and SYBR-I, the enriched E. coli O157:H7 was determined using multiparameter FCM analysis. With this method, the detection sensitivity was greatly improved due to the LMNPs enrichment and the signal amplification of the FSiNPs labelling method. Furthermore, the false positives caused by aggregates of FSiNPs conjugates and nonspecific binding of FSiNPs to background debris could be significantly decreased. This assay allowed the detection of E. coli O157:H7 in PB buffer at levels as low as 7 cells mL(-1). The total assay time including E. coli O157:H7 sample enrichment and detection was less than 4 h. An artificially contaminated bottled mineral water sample with a concentration of 6 cells mL(-1) can be detected by this method. It is believed that the proposed method will find wide applications in biomedical fields demanding higher sensitive bacterial identification.     

Synthesis of vitamin-selenium complex and its effects on proteins and tumor cells

A selenium-vitamin P complex (SEVP) was synthesized and its structure was determined by IR, LC-MS and (1)H NMR. Its biological effects on bovine serum albumin (BSA) and human colon carcinoma tumor cells were studied by molecular spectra, MTT and flow cytometry. The interaction of SEVP and BSA was discussed by fluorescence quenching method and F?rster non-radiation energy transfer theory. The thermodynamic parameters ΔH(θ), ΔG(θ), ΔS(θ) at different temperatures were calculated and the results indicate the interaction is an exothermic as well as entropy-driven process. Hydrogen bond and electrostatic force played major role in the reaction. The binding geometry and conformation changes of BSA were investigated by fluorescence probe technique and circular dichroism (CD) spectra. The effects of SEVP on human colon carcinoma tumor cells HT29 were tested by MTT method and flow cytometry (FCM). The MTT results show the proliferation of HT29 tumor cells were inhibited by SEVP and the inhibition was associated with dose and time. The FCM analysis disclosed SEVP interrupted the DNA synthesis of tumor cells at S phase.     

Single-cell detection of trans-splicing ribozyme in vivo activity

The Tetrahymena trans-splicing ribozyme can edit RNA in a sequence-specific manner, but its efficiency needs to be improved for any functional rescues. This communication describes a simple method that uses a bacterial enzyme beta-lactamase to report trans-splicing activity of Tetrahymena ribozyme in single living mammalian cells by fluorescence microscopy and flow cytometry. This enzyme-based single-cell detection method is highly sensitive and compatible with living cell flow cytometry, and should allow a cell-based systematic screening of a vast library of ribozymes for better trans-spliced ribozyme variants.     

Optimization of the preparation of glass-coated, dye-tagged metal nanoparticles as SERS substrates

Dye-tagged metal nanoparticles are of significant interest in SERS-based sensitive detection applications. Coating these particles in glass results in an inert spectral tag that can be used in applications such as flow cytometry with significant multiplexing potential. Maximizing the SERS signal obtainable from these particles requires care in partitioning available nanoparticle surface area (binding sites) between the SERS dyes and the functionalized silanes necessary for anchoring the glass coating. In this article, we use the metal-mediated fluorescence quenching of SERS dyes to measure surface areas occupied by both dyes and silanes and thus examine SERS intensities as a function of both dye and silane loading. Notably, we find that increased surface occupation by silane increases the aggregative power of added dye but that decreasing the silane coverage allows a greater surface concentration of dye. Both effects increase the SERS intensity, but obtaining the optimum SERS intensity will require balancing aggregation against surface dye concentration.     

Near-infrared laser-induced fluorescence detection in capillary electrophoresis

As capillary electrophoresis continues to focus on miniaturization, either through reducing column dimensions or situating entire electrophoresis systems on planar chips, advances in detection become necessary to meet the challenges posed by these electrophoresis platforms. The challenges result from the fact that miniaturization requires smaller load volumes, demanding highly sensitive detection. In addition, many times multiple targets must be analyzed simultaneously (multiplexed applications), further complicating detection. Near-infrared (NIR) fluorescence offers an attractive alternative to visible fluorescence for critical applications in capillary electrophoresis due to the impressive limits of detection that can be generated, in part resulting from the low background levels that are observed in the NIR. Advances in instrumentation and fluorogenic labels appropriate for NIR monitoring have led to a growing number of examples of the use of NIR fluorescence in capillary electrophoresis. In this review, we will cover instrumental components used to construct ultrasensitive NIR fluorescence detectors, including light sources and photon transducers. In addition, we will discuss various types of labeling dyes appropriate for NIR fluorescence and finally, we will present several applications that have used NIR fluorescence in capillary electrophoresis, especially for DNA sequencing and fragment analysis.     

A Covalent Reporter of β‐Lactamase Activity for Fluorescent Imaging and Rapid Screening of Antibiotic‐Resistant Bacteria

Bacterial resistance to antibiotics poses a great clinical challenge in fighting serious infectious diseases due to complicated resistant mechanisms and time‐consuming testing methods. Chemical reaction‐directed covalent labeling of resistance‐associated bacterial proteins in the context of a complicated environment offers great opportunity for the in‐depth understanding of the biological basis conferring drug resistance, and for the development of effective diagnostic approaches. In the present study, three fluorogenic reagents LRBL1–3 for resistant bacteria labeling have been designed and prepared on the basis of fluorescence resonance energy transfer (FRET). The hydrolyzed probes could act as reactive electrophiles to attach the enzyme, β‐lactamase, and thus facilitated the covalent labeling of drug resistant bacterial strains. SDS electrophoresis and MALDI‐TOF mass spectrometry characterization confirmed that these probes were sensitive and specific to β‐lactamase and could therefore serve for covalent and localized fluorescence labeling of the enzyme structure. Moreover, this β‐lactamase‐induced covalent labeling provides quantitative analysis of the resistant bacterial population (down to 5 %) by high resolution flow cytometry, and allows single‐cell detection and direct observation of bacterial enzyme activity in resistant pathogenic species. This approach offers great promise for clinical investigations and microbiological research.     

Quantification of Bacterial Metabolic Activities in the Gut by d-Amino Acid-Based In Vivo Labeling

Herein, we propose a metabolic d -amino acid-based labeling and in situ hybridization-facilitated (MeDabLISH) strategy for the quantitative analysis of the indigenous metabolic status of gut bacteria. The fluorescent d -amino acid (FDAA)-based labeling intensities of bacteria were found to highly correlate with their temporal and steady-state metabolic status. Then, after taxonomic identification of bacterial genera in the in vivo FDAA-labeled mouse gut microbiota, by corresponding fluorescence in situ hybridization (FISH) probes, the metabolic activities of different gut bacterial genera are quantified by flow cytometry, using FISH signals to differentiate genera and FDAA signals to indicate their basal metabolic levels. It was found that Gram-negative genera in the mouse microbiota have stronger metabolic activities during the daytime, and Gram-positive genera have higher activities at the night. Our strategy will be instrumental in deepening our understanding of the highly complex microbiota.     

Nanoparticle-encapsulated vis- and NIR-emissive fluorophores with different fluorescence decay kinetics for lifetime multiplexing

Bioanalytical, clinical, and security applications increasingly require simple, efficient, and versatile strategies to measure an ever increasing number of analytes or events in parallel in a broad variety of detection formats as well as in conjunction with chromatographic separation techniques or flow cytometry. An attractive alternative to common optical multiplexing and encoding methods utilizing spectral multiplexing/color encoding and intensity encoding is lifetime multiplexing, which relies on the discrimination between different fluorescent reporters based on their fluorescence decay kinetics. Here, we propose a platform of surface-functionalizable polymeric nanoparticles stained with fluorophores differing in their fluorescence lifetimes as a new multiplexing and encoding approach. Proof-of-concept measurements with different sets of lifetime-encoded polystyrene nanoparticles are presented, obtained via staining of preformed particles with visible (vis)- and near-infrared (NIR)-emissive organic dyes, which display very similar absorption and emission spectra to enable excitation and detection at the same wavelengths, yet sufficiently different fluorescence decay kinetics in suspension, thereby minimizing instrumentation costs. Data analysis was performed with a linear combination approach in the lifetime domain. Our results and first cell experiments with these reporter sets underline the suitability of our multiplexing strategy for the discrimination between and the quantification of different labels. This simple and versatile concept can be extended to all types of fluorophores, thereby expanding the accessible time scale, and can be used, e.g., for the design of labels and targeted probes for fluorescence assays and molecular imaging, cellular imaging studies, and barcoding applications, also in conjunction with spectral and intensity encoding.

Preparation of highly fluorescent magnetic nanoparticles for analytes-enrichment and subsequent biodetection

Bifunctional nanoparticles with highly fluorescence and decent magnetic properties have been widely used in biomedical application. In this study, highly fluorescent magnetic nanoparticles (FMNPs) with uniform size of ca. 40 nm are prepared by encapsulation of both magnetic nanoparticles (MNPs) and shell/core quantum dots (QDs) with well-designed shell structure/compositions into silica matrix via a one-pot reverse microemulsion approach. The spectral analysis shows that the FMNPs hold high fluorescent quantum yield (QY). The QYs and saturation magnetization of the FMNPs can be regulated by varying the ratio of the encapsulated QDs to MNPs. Moreover, the surface of the FMNPs can be modified to offer chemical groups for antibody conjugation for following use in target-enrichment and subsequent fluorescent detection. The in vitro immunofluorescence assay and flow cytometric analysis indicate that the bifunctional FMNPs-antibody bioconjugates are capable of target-enrichment, magnetic separation and can also be used as alternative fluorescent probes on flow cytometry for biodetection.     

Single channel layer, single sheath-flow inlet microfluidic flow cytometer with three-dimensional hydrodynamic focusing

Flow cytometry is a technique capable of optically characterizing biological particles in a high-throughput manner. In flow cytometry, three dimensional (3D) hydrodynamic focusing is critical for accurate and consistent measurements. Due to the advantages of microfluidic techniques, a number of microfluidic flow cytometers with 3D hydrodynamic focusing have been developed in recent decades. However, the existing devices consist of multiple layers of microfluidic channels and tedious fluidic interconnections. As a result, these devices often require complicated fabrication and professional operation. Consequently, the development of a robust and reliable microfluidic flow cytometer for practical biological applications is desired. This paper develops a microfluidic device with a single channel layer and single sheath-flow inlet capable of achieving 3D hydrodynamic focusing for flow cytometry. The sheath-flow stream is introduced perpendicular to the microfluidic channel to encircle the sample flow. In this paper, the flow fields are simulated using a computational fluidic dynamic (CFD) software, and the results show that the 3D hydrodynamic focusing can be successfully formed in the designed microfluidic device under proper flow conditions. The developed device is further characterized experimentally. First, confocal microscopy is exploited to investigate the flow fields. The resultant Z-stack confocal images show the cross-sectional view of 3D hydrodynamic with flow conditions that agree with the simulated ones. Furthermore, the flow cytometric detections of fluorescence beads are performed using the developed device with various flow rate combinations. The measurement results demonstrate that the device can achieve great detection performances, which are comparable to the conventional flow cytometer. In addition, the enumeration of fluorescence-labelled cells is also performed to show its practicality for biological applications. Consequently, the microfluidic flow cytometer developed in this paper provides a practical platform that can be used for routine analysis in biological laboratories. Additionally, the 3D hydrodynamic focusing channel design can also be applied to various applications that can advance the lab on a chip research.     

4-Azidocinnoline—Cinnoline-4-amine Pair as a New Fluorogenic and Fluorochromic Environment-Sensitive Probe

A new type of fluorogenic and fluorochromic probe based on the reduction of weakly fluorescent 4-azido-6-(4-cyanophenyl)cinnoline to the corresponding fluorescent cinnoline-4-amine was developed. We found that the fluorescence of 6-(4-cyanophenyl)cinnoline-4-amine is strongly affected by the nature of the solvent. The fluorogenic effect for the amine was detected in polar solvents with the strongest fluorescence increase in water. The environment-sensitive fluorogenic properties of cinnoline-4-amine in water were explained as a combination of two types of fluorescence mechanisms: aggregation-induced emission (AIE) and excited state intermolecular proton transfer (ESPT). The suitability of an azide–amine pair as a fluorogenic probe was tested using a HepG2 hepatic cancer cell line with detection by fluorescent microscopy, flow cytometry, and HPLC analysis of cells lysates. The results obtained confirm the possibility of the transformation of the azide to amine in cells and the potential applicability of the discovered fluorogenic and fluorochromic probe for different analytical and biological applications in aqueous medium.     

Quantification of Bacterial Metabolic Activities in the Gut by d‐Amino Acid‐Based In Vivo Labeling

Herein, we propose a metabolic d ‐amino acid‐based labeling and in situ hybridization‐facilitated (MeDabLISH) strategy for the quantitative analysis of the indigenous metabolic status of gut bacteria. The fluorescent d ‐amino acid (FDAA)‐based labeling intensities of bacteria were found to highly correlate with their temporal and steady‐state metabolic status. Then, after taxonomic identification of bacterial genera in the in vivo FDAA‐labeled mouse gut microbiota, by corresponding fluorescence in situ hybridization (FISH) probes, the metabolic activities of different gut bacterial genera are quantified by flow cytometry, using FISH signals to differentiate genera and FDAA signals to indicate their basal metabolic levels. It was found that Gram‐negative genera in the mouse microbiota have stronger metabolic activities during the daytime, and Gram‐positive genera have higher activities at the night. Our strategy will be instrumental in deepening our understanding of the highly complex microbiota.     

High-power blue/UV light-emitting diodes as excitation sources for sensitive detection

With advances in III-V nitride manufacturing processes, high-power light-emitting diode (LED) chips in the blue and UV wavelengths are now commercially available at reasonable cost and can be used as excitation sources in optical sensing. We describe the use of these high-power blue and UV LEDs for sensitive fluorescence detection, including chip-based flow cytometry, capillary electrophoresis (CE), and single-molecule imaging. By using a blue LED with a focusable power of approximately 40 mW as the excitation source for fluorescent beads, we demonstrate a simple chip-based bead sorter capable of enriching the concentration of green fluorescent beads from 63% to 95%. In CE experiments, we show that a mixture of analyte solution containing 30 nM 6-carboxyrhodamine 6G and 10 nM fluorescein can be separated and detected with excellent signal-to-noise ratio (approximately 17 for 10 nM fluorescein) using the collimated emission from a blue LED; the estimated mass detection limit was approximately 200 zmol for fluorescein. We also demonstrated ultrasensitive fluorescence imaging of single rhodamine 123 molecules and individual lambda-DNA molecules. At a small fraction of the cost of an Ar+ laser, high-power blue and UV LEDs are effective alternatives for lasers and arc lamps in fluorescence applications that demand portability, low cost, and convenience.     

细菌的核酸适配体筛选的研究进展

核酸适配体(aptamer)是通过指数富集配体系统进化技术(SELEX)筛选的能够以高亲和力和高特异性识别靶标分子或细胞的核糖核酸(RNA)和单链脱氧核糖核酸(ssDNA)。作为化学抗体,核酸适配体的制备和合成比抗体的成本更低。核酸适配体的靶标范围极其广泛,包括小分子、生物大分子、细菌和细胞等。针对细菌靶标筛选的适配体,目前主要应用于食品、医药和环境中的细菌检测。细菌的核酸适配体筛选可以通过离心法将菌体-适配体复合物与游离的适配体分离,并通过荧光成像、荧光光谱分析、流式细胞仪分选、DNA捕获元件、酶联适配体分析等方法表征适配体与靶标的相互作用。筛选出的适配体可结合生物、化学检测方法用于细菌检测。本文介绍了细菌适配体的筛选和表征方法以及基于适配体的检测方法的最新进展,分析了不同检测方法的利弊,并列出了2011~2015年筛选的细菌的核酸适配体。     

Large-volume sample stacking with polarity switching for the analysis of bacteria by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis has been utilized for the rapid analysis of bacteria under specific experimental conditions. In this work, a method of large-volume sample stacking with polarity switching was evaluated for the analysis of bacteria by capillary electrophoresis with laser-induced fluorescence detection in order to enhance the detection sensitivity. The results indicated that the proposed method is not only effective for the focusing of bacterial cells, but also for the separation of mixtures of bacteria. With the optimized conditions, an enhancement factor of around 60-fold was obtained when long sample plug (up to 39.6% of capillary volume) was injected. Moreover, with the help of such stacking method, single, sharp, intense peak with high efficiency was observed without multiple peaks attributable to irregular clusters and aggregates of bacterial cells. This simple stacking approach appears to be promising as a rapid sterility test in various fields of applications.     

Real-time monitoring for bioaerosols--flow cytometry

Bioaerosol detection in real time is an urgent civilian and military requirement. In this article, bioaerosol mass spectrometry, an instrument for real-time detection of bioaerosols using simultaneous measurement of particle aerodynamic size and intrinsic fluorescence, real-time qPCR, and FCM/FL were discussed. Although, challenging work remains to determine the interfering substances (e.g. particulates) of different environments, distinguish the specific species with specific probe, and overcome the high detection limit of FCM (10(4)-10(8) cells ml(-1)), literature reports suggested that FCM/FL has a great potential for real-time monitoring of bioaerosols.