Showing papers in "Analyst in 2019"

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification.

Abstract: Nucleic acid amplification has permeated every field in the life sciences since the introduction of the classic polymerase chain reaction (PCR) method in 1983. Yet, despite its fundamental reach, PCR has been constrained within the walls of a laboratory, due to its requirement for a sophisticated thermocycling machine, limiting external application in low-resource settings. New isothermal amplification strategies are seeking to break through traditional laboratory boundaries by providing nucleic acid replication at constant temperatures. Of these methods, recombinase polymerase amplification (RPA) is one of the fastest developing, experiencing rapid uptake and market, even though it was introduced comparatively late. Critically, RPA's technology potentiates highly accessible and sensitive nucleic acid amplification outside of laboratory, and even self-testing. Here we provide a comprehensive review of the equipment-free simplicity of RPA over its first decade of development. Our review includes key knowledge of RPA technology, such as its reaction components, mechanism, sensitivities and specificities, and distinctive detection methods. The review also provides know-how for developing RPA assays, and information about commercially available RPA reaction kits and accessories. We summarise critical RPA experimental tips and issues available through data mining the published literature, to assist researchers in mastering the RPA reaction. We also outline influential hotspots of RPA development, and conclude with outlooks for future development and implications for eclipsing PCR and further revolutionising the life sciences.

A review of methods for the detection of pathogenic microorganisms

Abstract: The testing and rapid detection of pathogenic organisms is a crucial protocol in the prevention and identification of crises related to health, safety and wellbeing. Pathogen detection has become one of the most challenging aspects in the food and water industries, because of the rapid spread of waterborne and foodborne diseases in the community and at significant costs. With the prospect of inevitable population growth, and an influx of tourism to certain water bodies testing will become a requirement to control and prevent possible outbreaks of potentially fatal illnesses. The legislation is already particularly rigorous in the food industry, where failure to detect pathogenic materials represents a catastrophic event, particularly for the elderly, very young or immune-compromised population types. In spite of the need and requirement for rapid analytical testing, conventional and standard bacterial detection assays may take up to seven days to yield a result. Given the advent of new technologies, biosensors, chemical knowledge and miniaturisation of instrumentation this timescale is not acceptable. This review presents an opportunity to fill a knowledge gap for an extremely important research area; discussing the main techniques, biology, chemistry, miniaturisation, sensing and the emerging state-of-the-art research and developments for detection of pathogens in food, water, blood and faecal samples.

A review of sorting, separation and isolation of cells and microbeads for biomedical applications: microfluidic approaches.

Abstract: Several biomedical analyses are performed on particular types of cells present in body samples or using functionalized microparticles. Success in such analyses depends on the ability to separate or isolate the target cells or microparticles from the rest of the sample. In conventional procedures, multiple pieces of equipment, such as centrifuges, magnets, and macroscale filters, are used for such purposes, which are time-consuming, associated with human error, and require several operational steps. In the past two decades, there has been a tendency to develop microfluidic techniques, so-called lab-on-a-chip, to miniaturize and automate these procedures. The processes used for the separation and isolation of the cells and microparticles are scaled down into a small microfluidic chip, requiring very small amounts of sample. Differences in the physical and biological properties of the target cells from the other components present in the sample are the key to the development of such microfluidic techniques. These techniques are categorized as filtration-, hydrodynamic-, dielectrophoretic-, acoustic- and magnetic-based methods. Here we review the microfluidic techniques developed for sorting, separation, and isolation of cells and microparticles for biomedical applications. The mechanisms behind such techniques are thoroughly explained and the applications in which these techniques have been adopted are reviewed.

Advances in mass spectrometry based single-cell metabolomics

Abstract: Metabolomics has grown into a prominent field contributing to the molecular understanding of complex biological processes in both health and disease. Furthermore, single-cells are known to display metabolic differences between seemingly homogeneous populations of cells. Single-cell metabolomics attempts to analyze many cellular metabolites from single cells to understand phenotypic heterogeneity, which is a significant challenge due to the low analyte abundances and limited sample volumes. Label-free metabolite detection can be achieved with mass spectrometry, which is capable of simultaneously analyzing hundreds of metabolites. Herein, we review the recent advances in mass spectrometry based single-cell metabolomics, highlighting the current state-of-the-art within the last three years, and identify the challenges to move the field forward.

Deep learning-based component identification for the Raman spectra of mixtures

Abstract: Raman spectroscopy is widely used as a fingerprint technique for molecular identification. However, Raman spectra contain molecular information from multiple components and interferences from noise and instrumentation. Thus, component identification using Raman spectra is still challenging, especially for mixtures. In this study, a novel approach entitled deep learning-based component identification (DeepCID) was proposed to solve this problem. Convolution neural network (CNN) models were established to predict the presence of components in mixtures. Comparative studies showed that DeepCID could learn spectral features and identify components in both simulated and real Raman spectral datasets of mixtures with higher accuracy and significantly lower false positive rates. In addition, DeepCID showed better sensitivity when compared with the logistic regression (LR) with L1-regularization, k-nearest neighbor (kNN), random forest (RF) and back propagation artificial neural network (BP-ANN) models for ternary mixture spectral datasets. In conclusion, DeepCID is a promising method for solving the component identification problem in the Raman spectra of mixtures.

Addressing the practicalities of anodic stripping voltammetry for heavy metal detection: a tutorial review.

Abstract: Anodic Stripping Voltammetry (ASV) has the capability to detect heavy metals at sub ppb-level with portable and cheap instrumentation making it ideal for in the field (at the source) analysis, however, commercial activity is surprisingly limited. The more commonly used liquid mercury electrodes are now obsolete due to toxicity concerns, and replacements are all based around solid electrodes, which come with their own challenges. This tutorial review aims to discuss the experimental practicalities of ASV, providing a clear overview of the issues for consideration, which can serve as a guide for anyone wanting to undertake analytical ASV. Choice of electrode material (with or without subsequent modification) and solution composition (pH, electrolyte, buffer) are important parameters, as well as an understanding of pH dependent metal speciation and possible intermetallic effects. Measurements made on model solutions often differ from those made on environmental samples with the latter containing organic matter, biological and inorganic species, which themselves can adsorb metal ions. Consideration should also be given to the method of solution collection and the sample container utilised. ASV can be a powerful tool to an analytical chemist, however optimisation for the application of interest is essential, which this review aims to help guide.

Recent advances in single cell manipulation and biochemical analysis on microfluidics

Abstract: Single cell analysis has become of great interest with unprecedented capabilities for the systematic investigation of cell-to-cell variation in large populations. Rapid and multi-parametric analysis of intercellular biomolecules at the single-cell level is imperative for the improvement of early disease diagnosis and personalized medicine. However, the small size of cells and the low concentration levels of target biomolecules are critical challenges for single cell analysis. In recent years, microfluidic platforms capable of handling small-volume fluid have been demonstrated to be powerful tools for single cell analysis. In addition, microfluidic techniques allow for precise control of the localized microenvironment, which yield more accurate outcomes. Many different microfluidic techniques have been greatly improved for highly efficient single-cell manipulation and highly sensitive detection over the past few decades. To date, microfluidics-based single cell analysis has become the hot research topic in this field. In this review, we particularly highlight the advances in this field during the past three years in the following three aspects: (1) microfluidic single cell manipulation based on microwells, micropatterns, droplets, traps and flow cytometric methods; (2) detection methods based on fluorescence, mass spectrometry, electrochemical, and polymerase chain reaction-based analysis; (3) applications in the fields of small molecule detection, protein analysis, multidrug resistance analysis, and single cell sequencing with droplet microfluidics. We also discuss future research opportunities by focusing on key performances of throughput, multiparametric target detection and data processing.

A graphene oxide/gold nanoparticle-based amplification method for SERS immunoassay of cardiac troponin I

Abstract: Cardiac troponin I (cTnI) was considered as the “gold standard” for acute myocardial infarction (AMI) diagnosis owing to its superior cardiac specificity for cardiac damage and showing little or no changes in patients with a skeletal muscle disease or trauma. Herein, a new signal amplification surface-enhanced Raman scattering (SERS) platform was developed for recognition and detection of cTnI by using gold nanoparticles (AuNPs), graphene oxide (GO) and magnetic beads (MB). Here, antibody/Raman reporter labeled AuNP–functionalized GO were employed as both SERS nanotags and signal amplification carriers. Monoclonal antibody modified MB were applied as the capture probe and separation agents. In the presence of cTnI, sandwich type immunocomplexes, “capture probe/target/SERS nanotags”, were formed through antibody–antigen–antibody interactions. Due to the strong SERS enhancement ability of the designed GO/AuNP complexes and a high binding chance between cTnI and the GO/AuNP complexes, the proposed SERS-based immunoassay could selectively detect cTnI with a high sensitivity (detection limit of 5 pg mL−1) and a good linearity was obtained in a range of 0.01–1000 ng mL−1. In addition, this method was also successfully applied for detecting cTnI in serum substitute media with a similar linear range. Furthermore, this strategy can be constructed with different kinds of antibodies and Raman reporters, and thus can be easily used for simultaneous detection of multiple biomarkers. Therefore, this proposed SERS-based signal amplification technique shows strong potential for the clinical diagnosis of AMI disease.

Magnetic dispersive micro-solid phase extraction with the CuO/ZnO@Fe3O4-CNTs nanocomposite sorbent for the rapid pre-concentration of chlorogenic acid in the medical extract of plants, food, and water samples.

Abstract: Herein, the CuO/ZnO@Fe3O4-carbon nanotubes (CNTs)-nanocomposite (NC), as a sorbent for magnetic dispersive micro-solid phase extraction, was developed for the determination of chlorogenic acid (CGA) in the medical extract of plants, food, and water samples in combination with high-performance liquid chromatography. The CuO/ZnO@Fe3O4-CNTs-NC was characterized by FESEM, EDS, XRD, TEM, BET, FT-IR, and VSM. Significant parameters (pH, temperature, eluent volume, vortex time, sonication time, CuO/ZnO@Fe3O4-CNTs-NC mass, and desorption time) that affected the extraction efficiency of CGA were optimized using Plackett-Burman as the screening design and the central composite design as the optimization strategy. Under the optimized conditions, the analytical parameters were obtained. The optimized method showed good linearity and a linear regression coefficient >0.9893. The enrichment factors ranged from 102.43 to 123.76 with the preconcentration factor of 60.0. The limits of detection (LOD) and the limits of quantification (LOQ) were 0.034-0.061 ng mL-1 and 0.114-0.202 ng mL-1, respectively. The method also reflected acceptable accuracy (from 94.07% to 109.7%) and the broad potential applications of CuO/ZnO@Fe3O4-CNTs-NC for the detection of CGA content in the medical extract of plants, food, and water samples.

Dual-template molecularly imprinted polymers for dispersive solid-phase extraction of fluoroquinolones in water samples coupled with high performance liquid chromatography

Abstract: Novel dual-template molecularly imprinted polymers (dt-MIPs) were prepared by simple and facile precipitation polymerization using norfloxacin (NOR) and enrofloxacin (ENR) as templates for simultaneous selective recognition and extraction of the two fluoroquinolones (FQs). The as-prepared dt-MIPs exhibited high adsorption capacity and excellent selectivity towards NOR and ENR. Several main parameters affecting the efficiency of dt-MIP based dispersive solid-phase extraction (DSPE) were systematically investigated, coupled with high performance liquid chromatography determination. Consequently, high enrichment factors of 71 and 61 were obtained for NOR and ENR respectively, and good linearity in the range of 1–200 μg L−1 was observed, with correlation coefficients (r) above 0.9977. The limits of detection and quantification for NOR were 0.22 and 0.67 μg L−1, respectively, and 0.36 and 0.98 μg L−1 for ENR. Satisfactory recoveries of the two FQs from spiked lake, sea and tap water samples at three concentration levels were attained in the range of 80.9–101.0% with relative standard deviations of 0.9–6.9%. The present study not only has great potential for applications in FQ determination, but will also enrich research into dual/multi-template imprinting.

Metal–organic framework-based fluorescent sensing of tetracycline-type antibiotics applicable to environmental and food analysis

Abstract: Antibiotics have been noted as an important class of emerging contaminants in the environment. Metal-organic frameworks (MOFs), which have been intensely investigated as a novel kind of sensing material, have been tentatively applied to the detection of antibiotics in recent years. In this work, a nanoscale MOF (In-sbdc) with a strong (quantum yield = 13%) and stable emission in water was synthesized. With its effective spectral overlap with tetracyclines, adsorption preconcentration and the usage of a masking agent, In-sbdc gave sensitive responses to a series of tetracycline antibiotics (tetracycline, chlorotetracycline and oxytetracycline) with detection limits of 0.28-0.30 μM, but another eight tested kinds of antibiotics did not cause a remarkable change in its emission (<10% of the response caused by an equal amount of tetracyclines). This MOF-based sensing system was successfully applied to tetracyclines detection in a series of actual water and food samples.

Recent advances in single-cell analysis by mass spectrometry

Abstract: Cells are the most basic structural units that play vital roles in the functioning of living organisms. Analysis of the chemical composition and content of a single cell plays a vital role in ensuring precise investigations of cellular metabolism, and is a crucial aspect of lipidomic and proteomic studies. In addition, structural knowledge provides a better understanding of cell behavior as well as the cellular and subcellular mechanisms. However, single-cell analysis can be very challenging due to the very small size of each cell as well as the large variety and extremely low concentrations of substances found in individual cells. On account of its high sensitivity and selectivity, mass spectrometry holds great promise as an effective technique for single-cell analysis. Numerous mass spectrometric techniques have been developed to elucidate the molecular profiles at the cellular level, including electrospray ionization mass spectrometry (ESI-MS), secondary ion mass spectrometry (SIMS), laser-based mass spectrometry and inductively coupled plasma mass spectrometry (ICP-MS). In this review, the recent advances in single-cell analysis by mass spectrometry are summarized. The strategies of different ionization modes to achieve single-cell analysis are classified and discussed in detail.

A new point-of-care test for the diagnosis of infectious diseases based on multiplex lateral flow immunoassays

Abstract: Infectious diseases are transmissible or communicable illnesses and can spread quickly in some areas and become epidemics. It is critical to quickly diagnose initial infections and prevent further spread through in vitro diagnosis. However, current detection strategies have exhibited a lack of balance with regard to accuracy, time consumption, and portability until recently (e.g. serology, culturing, molecular tests, etc.). Alternatively, many studies have focused on point-of-care testing (POCT), which combines simple, rapid, and exact on-site diagnostic platforms. Moreover, multiplex detectability is necessary for emergency treatment depending on the stage of the disease or interactional infections. The lateral flow assay (LFA) is the most popular diagnostic tool that meets the required standards for colorimetric assays. Here, we review lateral flow assays based on the immune reactions for the simultaneous diagnosis of infectious diseases as the POC test. The assays employed various forms and approaches in terms of the multiplexing level system for improving the sensitivity and specificity. We briefly describe the state-of-the-art infection diagnostic methods and published performances that have been classified into three categories based on the application forms of the lateral flow immunoassay. Also, we discuss further uses of LFA and other technologies for more effective infectious disease POCT.

Electrochemical sensors for rapid diagnosis of pathogens in real time

Abstract: Microbial infections remain the principal cause for high morbidity and mortality rates. While approximately 1400 human pathogens have been recognized, the majority of healthcare-associated infectious diseases are caused by only a few opportunistic pathogens (e.g., Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli), which are associated with increased antibiotic and antimicrobial resistance. Rapid detection, reliable identification and real-time monitoring of these pathogens remain not only a scientific problem but also a practical challenge of vast importance, especially in tailoring effective treatment strategies. Although the development of vaccinations and antibacterial drug treatments are the leading research, progress, and implementation of early warning, quantitative systems indicative of confirming pathogen presence are necessary. Over the years, various approaches, such as conventional culturing, straining, molecular methods (e.g., polymerase chain reaction and immunological assays), microscopy-based and mass spectrometry techniques, have been employed to identify and quantify pathogenic agents. While being sensitive in some cases, these procedures are costly, time-consuming, mostly qualitative, and are indirect detection methods. A great challenge is therefore to develop rapid, highly sensitive, specific devices with adequate figures of merit to corroborate the presence of microbes and enable dynamic real-time measurements of metabolism. As an alternative, electrochemical sensor platforms have been developed as powerful quantitative tools for label-free detection of infection-related biomarkers with high sensitivity. This minireview is focused on the latest electrochemical-based approaches for pathogen sensing, putting them into the context of standard sensing methods, such as cell culturing, mass spectrometry, and fluorescent-based approaches. Description of the latest, impactful electrochemical sensors for pathogen detection will be presented. Recent breakthroughs will be highlighted, including the use of micro- and nano-electrode arrays for real-time detection of bacteria in polymicrobial infections and microfluidic devices for pathogen separation analysis. We will conclude with perspectives and outlooks to understand shortcomings in designing future sensing schemes. The need for high sensitivity and selectivity, low-cost implementation, fast detection, and screening increases provides an impetus for further development in electrochemical detectors for microorganisms and biologically relevant targets.

Deep learning for FTIR histology: leveraging spatial and spectral features with convolutional neural networks

Abstract: Current methods for cancer detection rely on tissue biopsy, chemical labeling/staining, and examination of the tissue by a pathologist. Though these methods continue to remain the gold standard, they are non-quantitative and susceptible to human error. Fourier transform infrared (FTIR) spectroscopic imaging has shown potential as a quantitative alternative to traditional histology. However, identification of histological components requires reliable classification based on molecular spectra, which are susceptible to artifacts introduced by noise and scattering. Several tissue types, particularly in heterogeneous tissue regions, tend to confound traditional classification methods. Convolutional neural networks (CNNs) are the current state-of-the-art in image classification, providing the ability to learn spatial characteristics of images. In this paper, we demonstrate that CNNs with architectures designed to process both spectral and spatial information can significantly improve classifier performance over per-pixel spectral classification. We report classification results after applying CNNs to data from tissue microarrays (TMAs) to identify six major cellular and acellular constituents of tissue, namely adipocytes, blood, collagen, epithelium, necrosis, and myofibroblasts. Experimental results show that the use of spatial information in addition to the spectral information brings significant improvements in the classifier performance and allows classification of cellular subtypes, such as adipocytes, that exhibit minimal chemical information but have distinct spatial characteristics. This work demonstrates the application and efficiency of deep learning algorithms in improving the diagnostic techniques in clinical and research activities related to cancer.

Facile fabrication of fluorescent Fe-doped carbon quantum dots for dopamine sensing and bioimaging application

Abstract: In this paper, we have presented a novel strategy to fabricate Fe-doped carbon quantum dots (Fe-CQDs) for dopamine sensing applications. The Fe-CQDs are obtained by one step hydrothermal carbonization, using ethylenediamine tetraacetic acid salts and ferric nitrate as the carbon and iron source, which simultaneously incorporates Fe (dopamine-bonding site) and luminescent carbon quantum dots (fluorophores). The added dopamine containing catechol groups might form complexes with Fe ions (doped in CQDs) due to coordination. Subsequently, dopamine was oxidized to generate dopamine-quinone (a known potent electron acceptor) species by ambient O2. Thus, the coordination induced dopamine in proximity to the CQDs, which provided favourable electron acceptors in close proximity to the CQDs and produced high quenching efficiencies. Such fluorescence responses can be used for well quantifying dopamine in the range of 0.01-50 μM with a detection limit of 5 nM (S/N = 3). The proposed sensing system has been successfully used for the assay of dopamine in human urine samples. Preliminary cell image study indicates that the obtained Fe-CQDs possess high photostability and low cytotoxicity, which make them promising for biological applications.

Recent advances in nanomaterial-based electrochemical and optical sensing platforms for microRNA assays

Abstract: MicroRNA (MiRNA) plays a crucial role in biological cells to enable assessment of a cancer's development stage. Increasing evidence has shown that the accurate and sensitive detection of miRNA holds the key toward correct disease diagnosis. However, some characteristics of miRNAs, such as their short chains, low concentration, and similar sequences, make it difficult to detect miRNA in biological samples. Nanomaterials usually have good optical, electronic, and mechanical properties and therefore provide new possibilities for improving the performance of miRNA assays. Many different sorts of nanomaterials, including metal nanomaterials, carbon nanomaterials, quantum dots, and transition-metal dichalcogenides, have been used to construct optical and electrochemical assays for miRNA and have shown attractive results. This review describes recent efforts in the application of nanomaterials as sensing elements in electrochemical and optical miRNA assays. The analytical figures of merit of various methods for the detection of miRNA are compared in the present article. The current capabilities, limitations, and future challenges in miRNA detection and analysis based on nanomaterials are also addressed.

Microfluidic bioanalytical flow cells for biofilm studies: a review

Abstract: Bacterial biofilms are among the oldest and most prevalent multicellular life forms on Earth and are increasingly relevant in research areas related to industrial fouling, medicine and biotechnology. The main hurdles to obtaining definitive experimental results include time-varying biofilm properties, structural and chemical heterogeneity, and especially their strong sensitivity to environmental cues. Therefore, in addition to judicious choice of measurement tools, a well-designed biofilm study requires strict control over experimental conditions, more so than most chemical studies. Due to excellent control over a host of physiochemical parameters, microfluidic flow cells have become indispensable in microbiological studies. Not surprisingly, the number of lab-on-chip studies focusing on biofilms and other microbiological systems with expanded analytical capabilities has expanded rapidly in the past decade. In this paper, we comprehensively review the current state of microfluidic bioanalytical research applied to bacterial biofilms and offer a perspective on new approaches that are expected to drive continued advances in this field.

Paper-based SERS analysis with smartphones as Raman spectral analyzers

Abstract: SERS (Surface Enhanced Raman Spectroscopy) can realize fingerprint recognition of molecular samples with high detection accuracy and sensitivity. However, rapid and convenient measurement of the Raman spectra of analytes for a point-of-care test (POCT) has put forward a high demand for portable Raman spectrometers, as well as reliable SERS substrates. Hereby, we first utilize a smartphone as a miniaturized Raman spectral analyzer, which has the revolutionary advantages of a friendly human-machine interface, fast measurement time, and good sensitivity. Meanwhile, a paper-based SERS chip was prepared based on commonly used filter paper and silver nanoparticles (AgNP), which was successfully used to detect low concentrations of typical SERS analyte model molecules including rhodamine 6G and crystal violet. The current method of smartphone-based SERS spectroscopy as a POCT device will greatly promote the application of Raman technology in a variety of scenarios, such as safety inspections, pesticide residue detection, water pollution monitoring, and so on. Coupled with paper-based SERS chips with advantages of facile preparation, low cost and good reliability, the current work proves to have a great potential for industrial production and for meeting the vast marketing demand of Raman based POCT technology.

A Cd-MOF as a fluorescent probe for highly selective, sensitive and stable detection of antibiotics in water.

Abstract: Recently, the pollution and damage caused by antibiotics in water have aroused serious concerns. In this situation, it is extremely important to develop a highly effective approach to detect antibiotics in water. In this contribution, we built a Cd-MOF material with stable fluorescence properties, using bbi = 1,4-bis(2-methyl-imidazol-1-yl)butane and H2L = 1,2-phenylenediacetic acid as organic ligands and Cd(NO3)2·4H2O as the metal node. The highly selective response of this MOF probe to ceftriaxone sodium (an antibiotic) can reach up to the ppb level in water, along with a fast response time, acid and alkali resistance, and anti-interference ability.

New mass spectrometry technologies contributing towards comprehensive and high throughput omics analyses of single cells

Abstract: Mass-spectrometry based omics technologies - namely proteomics, metabolomics and lipidomics - have enabled the molecular level systems biology investigation of organisms in unprecedented detail. There has been increasing interest for gaining a thorough, functional understanding of the biological consequences associated with cellular heterogeneity in a wide variety of research areas such as developmental biology, precision medicine, cancer research and microbiome science. Recent advances in mass spectrometry (MS) instrumentation and sample handling strategies are quickly making comprehensive omics analyses of single cells feasible, but key breakthroughs are still required to push through remaining bottlenecks. In this review, we discuss the challenges faced by single cell MS-based omics analyses and highlight recent technological advances that collectively can contribute to comprehensive and high throughput omics analyses in single cells. We provide a vision of the potential of integrating pioneering technologies such as Structures for Lossless Ion Manipulations (SLIM) for improved sensitivity and resolution, novel peptide identification tactics and standards free metabolomics approaches for future applications in single cell analysis.

Fully inkjet-printed distance-based paper microfluidic devices for colorimetric calcium determination using ion-selective optodes

Abstract: Although the determination of calcium ions (Ca2+) is of high importance to monitor water hardness, currently available devices for on-site analysis suffer from a lack of user-friendliness and sensitivity. This work demonstrates fully inkjet-printed and low-cost microfluidic paper-based analytical devices (μPADs) for the simple naked-eye colorimetric determination of calcium ions (Ca2+) in drinking and tap water samples. The quantification of Ca2+ relies on visual readout of the length of a colour-changed detection channel modified with ionophore-doped ion-selective optode nanospheres (nano-optodes), eliminating the requirement of a scanner or a camera. All fabrication steps for deposition of assay reagents have been performed by means of a simple desktop thermal inkjet printer, which is expected to contribute to highly batch-to-batch reproducible device preparation. The detectable Ca2+ concentrations between 0.05 mmol L−1 and 5 mmol L−1 cover the range recommended by the International Organization for Standardization (0.05–2.5 mmol L−1) and the World Health Organization (WHO) guideline for Ca2+ quantification in drinking water (less than 5 mmol L−1). The lowest concentration of Ca2+ detectable by the naked eye was found to be 0.05 mmol L−1, which is below the value achieved with previously reported paper-based devices. μPAD quantified Ca2+ concentrations in tap or drinking waters were within 15% error of the results obtained with a classical complexometric titration. Hence, distance-based μPADs relying on nano-optodes are sensitive and reproducible tools for equipment-free on-site assaying of Ca2+ in real samples.

Synchrotron macro ATR-FTIR microspectroscopy for high-resolution chemical mapping of single cells.

Abstract: Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy has been used widely for probing the molecular properties of materials. Coupling a synchrotron infrared (IR) beam to an ATR element using a high numerical aperture (NA) microscope objective enhances the spatial resolution, relative to transmission or transflectance microspectroscopy, by a factor proportional to the refractive index (n) of the ATR element. This work presents the development of the synchrotron macro ATR-FTIR microspectroscopy at Australian Synchrotron Infrared Microspectroscopy (IRM) Beamline, and demonstrates that high quality FTIR chemical maps of single cells and tissues can be achieved at an enhanced spatial resolution. The so-called “hybrid” macro ATR-FTIR device was developed by modifying the cantilever arm of a standard Bruker macro ATR-FTIR unit to accept germanium (Ge) ATR elements with different facet sizes (i.e. 1 mm, 250 μm and 100 μm in diameter) suitable for different types of sample surfaces. We demonstrated the capability of the technique for high-resolution single cell analysis of malaria-infected red blood cells, individual neurons in a brain tissue and cellular structures of a Eucalyptus leaf. The ability to measure a range of samples from soft membranes to hard cell wall structures demonstrates the potential of the technique for high-resolution chemical mapping across a broad range of applications in biology, medicine and environmental science.

Deposition of CdTe quantum dots on microfluidic paper chips for rapid fluorescence detection of pesticide 2,4-D

Abstract: Rapid detection of pesticides in fruits is an ongoing challenge. The objective of the present study was to develop novel fluorescent microfluidic paper chips for specific recognition and sensitive detection of the pesticide 2,4-D through the electron-transfer-induced fluorescence quenching mechanism. CdTe quantum dots (QDs) were deposited onto cellulose paper (base material) to yield imprinted paper chips (paper@QDs@MIPs). This method allows the transferability of the molecularly imprinted fluorescence sensor from the liquid phase to the solid phase (paper base) for rapid and portable analysis. The resultant imprinted paper chips were effectively characterized, and they exhibited ideal ordered spatial network structure, chemical stability, and fluorescence property. The paper@QDs@MIPs showed that 2,4-D binding significantly reduced the fluorescence intensity within less than 18 min, and it achieved satisfactory linearity in the range of 0.83–100 μM and high detectability of 90 nM. The recognition specificity for 2,4-D relative to its analogues was shown, and the imprinting factor was 2.13. In addition, the recoveries of the spiked bean sprouts at three concentration levels ranged within 94.2–107.0%, with a relative standard deviation of less than 5.9%. Collectively, the device provided an effective platform for rapid recognition, convenience, and detection of trace food pollutants in complex matrices, thereby ensuring food safety and further promoting surface imprinting studies.

Dispersive micro-solid phase extraction based on Fe3O4@SiO2@Ti-MOF as a magnetic nanocomposite sorbent for the trace analysis of caffeic acid in the medical extracts of plants and water samples prior to HPLC-UV analysis

Abstract: In the present work, a simple and rapid method, namely magnetic dispersive micro-solid phase extraction (MD-μ-SPE), was developed using magnetic Fe3O4@SiO2@Ti-MOF (metal-organic framework)-nanocomposites (NCs) as a viable and efficient sorbent to pre-concentrate and assess caffeic acid (CA) in the medical extracts of plants and water samples. The Fe3O4@SiO2@Ti-MOF-NCs were characterized by various techniques, including FESEM, EDX, FTIR, BET, XRD and VSM. Following the extraction process, HPLC was employed to quantify CA. The effects of different parameters on the MD-μ-SPE method were fully investigated by the Plackett-Burman (PBD) and central composite design (CCD). The highest extraction percentage (99.76%) was obtained under different conditions: pH 4.8, 9 mg of Fe3O4@SiO2@Ti-MOF-NCs, sonication for 5 min, and an eluent solvent of 240 μL. Under optimum conditions, the limits of detection (LODs) and the limits of quantitation (LOQs) were obtained in the range of 0.016-0.021 ng mL-1 and 0.052-0.068 ng mL-1, respectively. The reproducibility and repeatability (RSDs, n = 5) were in the range of 3.65-8.66% and 1.84-5.54%, respectively. Overall, the proposed method was successfully used to determine CA in the medical extracts of plants and water samples with favorable recoveries.

Advancing single-cell proteomics and metabolomics with microfluidic technologies

Abstract: Recent advances in single-cell analysis have unraveled substantial heterogeneity among seemingly identical cells at genomic and transcriptomic levels. These discoveries have urged scientists to develop new tools that are capable of investigating single cells from a broader set of “omics”. Proteomics and metabolomics, for instance, are of particular interest as they are closely correlated with a dynamic picture of cellular behaviors and phenotypic identities. The development of such tools requires highly efficient isolation and processing of a large number of individual cells, where techniques such as microfluidics are extremely useful. Here, we review the recent advances in single-cell proteomics and metabolomics, with a focus on microfluidics-based platforms. We highlight a vast array of emerging microfluidic formats for single-cell isolation and manipulation, and how the state-of-the-art analytical tools are coupled with such platforms for proteomic and metabolomic profiling.

Advances in nanoplasmonic biosensors for clinical applications.

Abstract: Biomarkers are unquestionable biological indicators for diagnosis and therapeutic interventions providing appropriate classification of a wide range of health disorders and risk factors. Nonetheless, the detection and quantification of biomarkers need to be tested with sufficient reliability by robust analytical methods in order to assure clinical performance in health care settings. Since the analytical performance is determined by the sensitivity and specificity of the method employed, techniques have been intensively refined in order to avoid the misinterpretation of results and undesirable bias. Although biomarkers can be detected with the existing analytical techniques, to reproducibly quantify them in decentralized settings or remote locations with the required accuracy is still a challenge. Currently, only a few point-of-care devices for biomarker evaluation are commercially available. Thus, more focused research efforts are needed to overcome these limitations in order to provide universal patient-centered care platforms. To this end, plasmonic biosensors can be conveniently used as portable diagnostic devices for attaining timely and cost-effective clinical outcomes. The development of enhanced performance based on nanoplasmonics technology opens the way for sensor miniaturization, multiplexing and point of care testing. This review covers recent advances and applications of plasmonic and nanoplasmonic biosensors intended for biomarker diagnosis in clinical practice, including cancer, cardiovascular and neurodegenerative diseases. The review specially focuses on: (i) recent progress in plasmonics development including the design of singular nanostructured surfaces, (ii) novel chemical functionalization strategies for the appropriate incorporation of bioreceptors and (iii) plasmonic applications as real operative devices in the clinical field. Future prospects in the use of nanoplasmonic sensor platforms for personalised quantification and management of biomarkers directly in body fluids will also be discussed.

High throughput screening of complex biological samples with mass spectrometry – from bulk measurements to single cell analysis

Abstract: High throughput screening (HTS) of molecular analytes is in high demand from and implemented in many areas of chemistry, medicine and industrial biotechnology including the discovery of biomarkers and the development of new chemical entities. Despite its prevalence, technical challenges remain in many of the new application areas of HTS which require rapid results from complex mixtures, for example in: screening biotransformations; targeted metabolomics; and in locating drugs and/or metabolites in biological matrices. Common to all of these are lengthy and costly sample preparation stages, involving recovery from cell cultures, extractions followed by low throughput LC-MS/MS methods or specific fluorescence measurements. In the latter the target molecules need to be inherently fluorescent or to include a fluorescent label or tag which can adversely influence a cellular system. Direct infusion mass spectrometry coupled with robotic sample infusion is a viable contender for information rich HTS with sub-second analysis times, and recent developments in ambient ionisation have heralded a new era where screening can be performed on crude cell lysates or even from live cells. Besides commercially available technologies such as RapidFire, Acoustic Mist Ionisation, and the TriVersa ChipMate there are promising new developments from academic groups. Novel applications using desorption electrospray ionisation, microfluidics, rapid LC-separation and 'one cell' direct infusion methods offer much potential for increasing throughput from 'messy' complex samples and for significantly reducing the amount of material that needs to be analysed. Here we review recent advances in HTS coupled with MS with an emphasis on methods that reduce or remove all sample preparation and will facilitate single cell screening approaches.

Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping.

Abstract: Porous graphitized carbon (PGC) based chromatography achieves high-resolution separation of glycan structures released from glycoproteins. This approach is especially valuable when resolving structurally similar isomers and for discovery of novel and/or sample-specific glycan structures. However, the implementation of PGC-based separations in glycomics studies has been limited because system-independent retention values have not been established to normalize technical variation. To address this limitation, this study combined the use of hydrolyzed dextran as an internal standard and Skyline software for post-acquisition normalization to reduce retention time and peak area technical variation in PGC-based glycan analyses. This approach allowed assignment of system-independent retention values that are applicable to typical PGC-based glycan separations and supported the construction of a library containing >300 PGC-separated glycan structures with normalized glucose unit (GU) retention values. To enable the automation of this normalization method, a spectral MS/MS library was developed of the dextran ladder, achieving confident discrimination against isomeric glycans. The utility of this approach is demonstrated in two ways. First, to inform the search space for bioinformatically predicted but unobserved glycan structures, predictive models for two structural modifications, core-fucosylation and bisecting GlcNAc, were developed based on the GU library. Second, the applicability of this method for the analysis of complex biological samples is evidenced by the ability to discriminate between cell culture and tissue sample types by the normalized intensity of N-glycan structures alone. Overall, the methods and data described here are expected to support the future development of more automated approaches to glycan identification and quantitation.

Enzyme-triggered in situ formation of Ag nanoparticles with oxidase-mimicking activity for amplified detection of alkaline phosphatase activity

Abstract: Given that alkaline phosphatase (ALP) is an important biomarker for many diseases, monitoring of its activity turns to be of great significance for related disease diagnosis and treatment. Herein, we report a new colorimetric assay based on the enzyme-triggered in situ formation of Ag nanoparticles (NPs) with high oxidase-mimicking activity for ALP activity detection. ALP first hydrolyzes the ascorbic acid phosphate (AAP) substrate to produce ascorbic acid (AA); the produced AA with strong reducing capacity then transforms Ag+ into Ag NPs; compared with the Ag+ precursor, the in situ formed Ag NPs have much higher oxidase-like activity to catalyze the 3,3′,5,5′-tetramethylbenzidine (TMB) color reaction mediated by dissolved O2 at neutral pH. On the basis of this principle, amplified colorimetric detection of ALP activity with a linear scope of 0.15–5 U L−1 and a limit of detection down to 0.037 U L−1 was realized. In addition, our assay exhibited specific response toward ALP against other biological enzymes and species. Accurate and reliable determination of ALP activity in human plasma was also demonstrated by our assay, suggesting its great potential as a facile and efficient tool for monitoring of ALP activity in clinical practice.