Integration of polymeric membranes with microfluidic networks for bioanalytical applications.

The concept of microfluidics has significantly influenced the design and the implementation of modern bioanalytical systems due to the fact that these miniaturized devices can handle and manipulate samples in a much more efficient way than conventional instruments. In an analogy to the development of microelectronics, increasingly sophisticated devices with greater functionalities have become one of the major goals being pursued in the area of micrototal analysis systems. The incorporation of polymeric membranes into microfluidic networks has therefore been employed in an effort to enhance the functionalities of these microfabricated devices. These commercially available membranes are porous, flexible, mechanically robust and compatible with plastic microfluidic networks. The large surface area-to-volume ratio of porous membrane media is particularly important for achieving rapid buffer exchange during microdialysis and obtaining ultrahigh concentration of adsorbed enzymes for various biochemical reactions. Furthermore, the membrane pore diameter in the sub-microm range eliminates the constraints of diffusional mass-transfer resistance for performing chiral separation using adsorbed protein as the chiral stationary phase. A review on the recent advancement in the integration of polymeric membranes with microfluidic networks is presented for their widespread applications in bioanalytical chemistry.     

In-channel atom-transfer radical polymerization of thermoset polyester microfluidic devices for bioanalytical applications

A new technique for polymer microchannel surface modification, called in-channel atom-transfer radical polymerization, has been developed and applied in the surface derivatization of thermoset polyester (TPE) microdevices with poly(ethylene glycol) (PEG). X-ray photoelectron spectroscopy, electroosmotic flow (EOF), and contact angle measurements indicate that PEG has been grafted on the TPE surface. Moreover, PEG-modified microchannels have much lower and more pH-stable EOF, more hydrophilic surfaces and reduced nonspecific protein adsorption. Capillary electrophoresis separation of amino acid and peptide mixtures in these PEG-modified TPE microchips had good reproducibility. Phosducin-like protein and phosphorylated phosducin-like protein were also separated to measure the phosphorylation efficiency. Our results indicate that PEG-grafted TPE microchips have broad potential application in biomolecular analysis.     

Molecular beacons for bioanalytical applications

Molecular beacons (MBs) are hairpin-shaped oligonucleotides that contain both fluorophore and quencher moieties. They act like switches and are normally in a closed state, when the fluorophore and the quencher are brought together to turn "off" the fluorescence. When prompted to undergo conformational changes that open the hairpin structure, the fluorophore and the quencher are separated, and fluorescence is turned "on." This Education will outline the principles of MBs and discuss recent bioanalytical applications of these probes for in vitro RNA and DNA monitoring, biosensors and biochips, real-time monitoring of genes and gene expression in living systems, as well as the next generation of MBs for studies on proteins, the MB aptamers. These important applications have shown that MBs hold great potential in genomics and proteomics where real-time molecular recognition with high sensitivity and excellent specificity is critical.     

Enzyme-functionalized mesoporous silica for bioanalytical applications

The unique properties of mesoporous silica materials (MPs) have attracted substantial interest for use as enzyme-immobilization matrices. These features include high surface area, chemical, thermal, and mechanical stability, highly uniform pore distribution and tunable pore size, high adsorption capacity, and an ordered porous network for free diffusion of substrates and reaction products. Research demonstrated that enzymes encapsulated or entrapped in MPs retain their biocatalytic activity and are more stable than enzymes in solution. This review discusses recent advances in the study and use of mesoporous silica for enzyme immobilization and application in biosensor technology. Different types of MPs, their morphological and structural characteristics, and strategies used for their functionalization with enzymes are discussed. Finally, prospective and potential benefits of these materials for bioanalytical applications and biosensor technology are also presented. Figure Enzyme-functionalized mesoporous silica fibers and their integration in a biosensor design. The immobilization process takes place essentially in the silica micropores.     

Analytical and bioanalytical applications of carbon dots

Carbon dots (CDs) comprise a recently discovered class of strongly fluorescent, emission-color-tuning and non-blinking nanoparticles with great analytical and bioanalytical potential. Raw CDs can be obtained by laser ablation or electrochemical exfoliation of graphite, from soot, or thermal carbonization, acid dehydration or ultrasonic treatment of molecular precursors. Passivation of raw CDs makes them fluorescent and their functionalization confers reactivity towards selected targets. CDs can be excited by single-photon (ultraviolet or near-ultraviolet) and multi-photon (red or near-infrared) excitation, and their luminescence properties are due to surface defects. CDs are being proposed as bioimaging probes because they comprise non-toxic elements and are biocompatible. Passivated and functionalized CDs can be made to sense pH, metal ions and molecular substances.     

Exploring bioanalytical applications of assisted protein reassembly

Reassembly of protein from its peptide fragments is a technique that can have many applications in the bioanalytical field. Typically, a reporter protein fragmented into its two peptides is employed as a label in this study. This fragments of peptide can reassemble yielding an active functional reporter. This reassembly of the protein can be assisted by non-covalently interacting peptides or proteins, which are attached to the fragmented reporter. This technique has been employed in several applications including study of protein–protein interactions, antibody screening, immunoassays, and high-throughput screening. This review focuses on different reporters employed in the study of reassembly of proteins and applications of this strategy in bioanalysis.     

DNA-based bioanalytical microsystems for handheld device applications

This article reviews and highlights the current development of DNA-based bioanalytical microsystems for point-of-care diagnostics and on-site monitoring of food and water. Recent progresses in the miniaturization of various biological processing steps for the sample preparation, DNA amplification (polymerase chain reaction), and product detection are delineated in detail. Product detection approaches utilizing “portable” detection signals and electrochemistry-based methods are emphasized in this work. The strategies and challenges for the integration of individual processing module on the same chip are discussed.     

Development of an iridium-based pH sensor for bioanalytical applications

A new iridium-based planar pH sensor for bioanalytical purposes is introduced. The fabrication of the sensor was carried out by a two-stage coating process of different iridium solutions on a platinum thick film surface. The pH response behaviour and the Nernstian characteristics of the double-layer electrode exhibited better results than the single iridium depositions. An almost theoretical Nernstian slope could be obtained as well as a pH response time of about 3 to 5 min in a pH range of 4.01 to 9.18. Furthermore, a biofilm growth of different microorganisms onto the iridium-coated electrodes could be achieved. Afterwards, the viability of the microorganisms was demonstrated via cell plating studies.     

Risk-based approach for the transfer of quantitative methods: bioanalytical applications

The transfer of analytical methods from a sending laboratory to a receiving one requires to guarantee that this last laboratory will obtain accurate results. Undeniably method transfer is the ultimate step before routine implementation of the method at the receiving site. The conventional statistical approaches generally used in this domain which analyze separately the trueness and precision characteristics of the receiver do not achieve this. Therefore, this paper aims first at demonstrating the applicability of two recent statistical approaches using total error-based criterion and taking into account the uncertainty of the true value estimate of the sending laboratory, to the transfer of bioanalytical methods. To achieve this, they were successfully applied to the transfer of two fully automated liquid chromatographic method coupled on-line to solid-phase extraction. The first one was dedicated to the determination of three catecholamines in human urine using electrochemical detection, and the second one to the quantitation of N-methyl-laudanosine in plasma using fluorescence detection. Secondly, a risk-based evaluation is made in order to understand why classical statistical approaches are not sufficient to provide the guarantees that the analytical method will give most of the time accurate results during its routine use. Finally, some recommendations for the transfer studies are proposed.     

Silicon couplers for microfluidic applications

Several types of silicon fluidic coupler have been designed, fabricated, and tested to facilitate external connections to MEMS (microelectromechanical systems) fluidic devices. By using both bulk micromachining and DRIE (deep reactive ion-etching) techniques, couplers of different geometry have been produced for use with any standard MEMS fluidic port. In addition, couplers are easily modified to accommodate any arbitrary fluidic port geometry. For ease of use, these couplers interface with PEEK (polyetheretherketone) and fused-silica capillary tubing, both of which are commonly used in HPLC (high-performance liquid chromatography) systems and are supported by a wide range of plumbing products. Coupler performance was evaluated and an operating range of at least 0-8,963 kPa (0-1,300 psig) is attainable.     

Bacterial spores as platforms for bioanalytical and biomedical applications

Genetically engineered bacteria-based sensing systems have been employed in a variety of analyses because of their selectivity, sensitivity, and ease of use. These systems, however, have found limited applications in the field because of the inability of bacteria to survive long term, especially under extreme environmental conditions. In nature, certain bacteria, such as those from Clostridium and Bacillus genera, when exposed to threatening environmental conditions are capable of cocooning themselves into a vegetative state known as spores. To overcome the aforementioned limitation of bacterial sensing systems, the use of microorganisms capable of sporulation has recently been proposed. The ability of spores to endow bacteria-based sensing systems with long lives, along with their ability to cycle between the vegetative spore state and the germinated living cell, contributes to their attractiveness as vehicles for cell-based biosensors. An additional application where spores have shown promise is in surface display systems. In that regard, spores expressing certain enzymes, proteins, or peptides on their surface have been presented as a stable, simple, and safe new tool for the biospecific recognition of target analytes, the biocatalytic production of chemicals, and the delivery of biomolecules of pharmaceutical relevance. This review focuses on the application of spores as a packaging method for whole-cell biosensors, surface display of recombinant proteins on spores for bioanalytical and biotechnological applications, and the use of spores as vehicles for vaccines and therapeutic agents.     

Frontal photopolymerization for microfluidic applications

Frontal photopolymerization (FPP) offers numerous advantages for the rapid prototyping of microfluidic devices. Quantitative utilization of this method, however, requires a control of the vertical dimensions of the patterned resist material. To address this fundamental problem, we study the ultraviolet (UV) photopolymerization of a series of multifunctional thiolene resists through a combination of experiments and analytical modeling of the polymerization fronts. We describe this nonlinear spatio-temporal growth process in terms of a "minimal" model involving an order parameter phi(x, t) characterizing the extent of monomer-to-polymer conversion, the optical attenuation T(x, t), and the solid front position h(t). The latter exhibits an induction time (or equivalent critical UV dose) characterizing the onset of frontal propagation. We also observe a novel transition between two logarithmic rates of growth, determined by the Beer-Lambert attenuation constants mu(0) and mu(infinity) of the monomer and fully polymerized material, respectively. The measured frontal kinetics and optical transmission of the thiolene resist materials are consistent with our photopolymerization model, exhibiting both "photodarkening" and "photoinvariant" polymerization. This is apparently the first observation of photodarkening reported in FPP. On the basis of these results, multilevel fluidic devices with controlled height are readily fabricated with modulated illumination. A representative two-level microfluidic device, incorporating a chaotic mixer, a T junction, and a series of controlled flow constrictions, illustrates the practical versatility of this fabrication method.     

Electrochemical techniques for microfluidic applications

Electrochemical principles provide key techniques to promote the construction of bio/chemical microsystems of the next generation. There is a wealth of technology for the microfabrication of bio/chemical sensors. In addition, microfluidic transport in a network of flow channels, pH regulation, and automatic switching can be realized by electrochemical principles. Since the basic components of the devices are electrode patterns, the integration of different components is easily achieved. With these techniques, bio/chemical assays that require the exchange of solutions can be conducted on a chip. Furthermore, autonomous microanalysis systems that can carry out necessary procedures are beginning to be realized. In this article, techniques developed in our group will be comprehensively introduced.     

Bioconjugated quantum dots as fluorescent probes for bioanalytical applications

Quantum dots (QDs) are inorganic semiconductor nanocrystals that have unique optoelectronic properties responsible for bringing together multidisciplinary research to impel their potential bioanalytical applications. In recent years, the many remarkable optical properties of QDs have been combined with the ability to make them increasingly biocompatible and specific to the target. With this great development, QDs hold particular promise as the next generation of fluorescent probes. This review describes the developments in functionalizing QDs making use of different bioconjugation and capping approaches. The progress offered by QDs is evidenced by examples on QD-based biosensing, biolabeling, and delivery of therapeutic agents. In the near future, QD technology still faces some challenges towards the envisioned broad bioanalytical purposes.      

Recent advances in SALDI-MS techniques and their chemical and bioanalytical applications

Although laser desorption mass spectrometry was introduced in the 1960s, the potential of laser mass spectrometry was not realised until the introduction of matrix-assisted laser desorption/ionisation (MALDI) in the 1980s. The technique relies on light-absorbing compounds called matrices that are co-crystallised with the analyte to achieve high ionisation and desorption efficiencies. MALDI offers a lot of advantages and is an indispensable tool in macromolecule analysis. However, the presence of the matrix also produces a high chemical background in the region below m/z 700 in the mass spectrum. Surface-assisted laser desorption/ionisation (SALDI) substitutes the chemical matrix of MALDI for an active surface, which means that matrix interference can be eliminated. SALDI mass spectrometry has evolved in recent years into a technique with great potential to provide insight into many of the challenges faced in modern research, including the growing interest in “omics” and the demands of pharmaceutical science. A great variety of materials have been reported to work in SALDI. Examples include a number of nanomaterials and surfaces. The unique properties of nanomaterials greatly facilitate analyte desorption and ionisation. This article reviews recent advances made in relation to carbon- and semiconductor-based SALDI strategies. Examples of their environmental, chemical and biomedical applications are discussed with the aim of highlighting progression in the field and the robustness of the technique, as well as to evaluate the strengths and weaknesses of individual approaches. In addition, this article describes the physical and chemical processes involved in SALDI and explains how the unique physical and electronic properties of nanostructured surfaces allow them to substitute for the matrix in energy transfer processes.     

Calorimetric sensing in bioanalytical chemistry: Principles, applications and trends

Calorimetry has shown great potential in bioanalytical chemistry as most biochemical processes involve a change in enthalpy. Two types of approach have been developed: (1) adiabatic calorimetry, which relies on the absence of heat exchange between the reaction vessel and the external environment, and (2) heat conduction calorimetry, involving measurement of the heat transferred from the vessel to a surrounding heat sink. Both principles, with their respective advantages and drawbacks, have been applied to microcalorimetry for the analysis of (bio)chemical compounds. Immobilization of the biomaterial in the vicinity of, or directly onto a small temperature or heat sensitive transducer has led to the concept of a calorimetric biosensor. In comparison to the traditional calorimeter, the calorimetric biosensor is better suited to continuous monitoring and size reduction. This simplified but sensitive device is expected to solve numerous problems in various fields of analytical chemistry.     

Voltammetric behavior of mammalian tumor cells and bioanalytical applications in cell metabolism

The voltammetric behavior of human mammalian cells was studied by choosing human leukemia cells (HL60) and human erythroleukemia cells (HEL). The voltammetric response of the cells was found having relation with cell metabolic viability in culture course. For example, the fluctuations of peak currents of HL60 were parallel with the nutrients replenished or not, which can reflect cell health state; the voltammetric response of HL60 regulated by the anti-metabolic drug 5F-Uriacil (5F-U) in culture course behaved in a much decreased manner, by which a voltammetric method for evaluating cytotoxicity is proposed. In this paper, the relation between HEL cell metabolism and the activation of receptor Mpl by its ligand TPO was also studied. Moreover, the mechanism of cell voltammetric behavior was discussed.