Measurement of neuropeptides in clinical samples using chip-based immunoaffinity capillary electrophoresis

The current interest in micro-fabrication has extended to the clinical arena where there is a growing lobby for promoting these for point-of-care purposes. The advantages of such devices are their relative speed of analysis, lower reagent costs, and their application to clinical screening and diagnosis. Two chip-based capillary electrophoresis systems have been designed and their performance evaluated for rapidly measuring the concentrations of inflammatory neuropeptides in tissue fluids of patients with neuropeptide-associated muscle pain. Both chips were manufactured to fit a commercially available chip electrophoresis system. One chip was designed to perform electrokinetic flow immunoassays while the other utilized an immunoaffinity port, containing an array of immobilized antibodies, to capture the analytes of interest. Comparison of the results to commercially available high-sensitivity immunoassays demonstrated that both chip-based systems could provide a relatively fast, accurate procedure for studying inflammatory biomarkers in complex biological fluids. However, the immunoaffinity capture system proved the superior of the two chips. Using this system, twelve different inflammation-associated mediators could be determined in approximately 2 min as compared to 30 min when using the flow immunoassay chip. With the ever-expanding array of antibodies that are commercially available, this chip-based system can be applied to a wide variety of different analyses.     

Automated sample preparation method for suspension arrays using renewable surface separations with multiplexed flow cytometry fluorescence detection

In this paper, we describe a new method of automated sample preparation for multiplexed biological analysis systems that use flow cytometry fluorescence detection. In this approach, color-encoded microspheres derivatized to capture particular biomolecules are temporarily trapped in a renewable surface separation column to enable perfusion with sample and reagents prior to delivery to the detector. This method provides for separation of the biomolecules of interest from other sample matrix components as well as from labeling solutions. After sample preparation, the beads can be released from the renewable surface column and delivered to a flow cytometer for direct on-bead analysis one bead at a time. Using mixtures of color-encoded beads derivatized for various analytes yields suspension arrays for multiplexed analysis. Development of this approach required a new technique for automated capture and release of the color-encoded microspheres within a fluidic system. We developed a method for forming a renewable filter and demonstrate its use for capturing microspheres that are too small to be easily captured in previous flow cells for renewable separation columns. The renewable filter is created by first trapping larger beads in the flow cell, and then smaller beads are captured either within or on top of the bed of larger beads. Both the selective microspheres and filter bed are automatically emplaced and discarded for each sample. A renewable filter created with 19.9 μm beads was used to trap 5.6 μm optically encoded beads with trapping efficiencies of 99%. The larger beads forming the renewable filter did not interfere with the detection of color-encoded 5.6 μm beads by the flow cytometer fluorescence detector. The use of this method was demonstrated with model reactions for a variety of bioanalytical assay types including a one-step capture of a biotinylated label on Lumavidin beads, a two-step sandwich immunoassay, and a one-step DNA binding assay. A preliminary demonstration of multiplexed detection of two analytes using color-encoded beads was also demonstrated. The renewable filter for creating separation columns containing optically encoded beads provides a general platform for coupling renewable surface methods for sample preparation and analyte labeling with flow cytometry detectors for suspension array multiplexed analyses.     

Capillary isoelectric focusing-mass spectrometry: analysis of protein mixtures from human body fluids

Isoelectric focusing within a fused silica capillary (cIEF) has proved to be a powerful and practical method for high-resolution separation of analytes from complex biological mixtures. This technique overcomes many of the problems of isoelectric focusing within slab gel media. However current cIEF systems commonly utilize UV detection which limits the detail of analyte structural information that is obtained during analysis. The use of mass spectrometry (MS) as the detection system provides much greater structural information about the detected analytes allowing accurate relative molecular mass (M(r)) determination for proteins and polypeptides. We have constructed a cIEF-MS interface and compared the separation of standard proteins analyzed by cIEF-UV with cIEF-MS. This allowed rapid optimization of the cIEF-MS system performance. Further we have demonstrated the use of MS as a detection system provides accurate M(r) information and can provide analyte modification details. These factors increase the likelihood of absolute identification for physiological proteins within complex in vivo-derived mixtures. To demonstrate the value of cIEF-MS in such analyses we have undertaken an examination of cerebrospinal fluid (CSF), and tentatively identified a number of constituent proteins. We have also analyzed whole blood from control and diabetic patients. We show that glycated alpha- and beta- chains of hemoglobin are found in almost equal abundance in diabetic patient blood. From these results we suggest cIEF-MS is an efficient and useful tool for the separation and examination of in vivo-derived analytes within physiological fluids.     

Microfluidic fabrication of SERS-active microspheres for molecular detection

In this paper, we demonstrated a microfluidic system for fabricating microspheres with hierarchical surface nanopatterns for molecular detection based on surface-enhanced Raman scattering (SERS). Briefly, a photocurable silica suspension was emulsified into monodisperse droplets using a microfluidic device composed of two coaxial glass capillaries. The silica particles in each droplet protruded through the interface and spontaneously formed a hexagonal array. After polymerization of the droplets, we selectively decorated the exposed areas of the silica particles with silver nanoparticles through electroless deposition. The resulting hierarchically-structured microspheres showed high sensitivity and fast binding kinetics in molecular detection based on SERS, owing to the dense array of hot spots on each microsphere and high mobility of the microspheres, respectively. Notably, the SERS signals from molecules adsorbed on the microspheres could be detected in both the dried and suspension states. In addition, we demonstrated that the SERS-active microspheres can be functionalized into structural colored or magnetoresponsive microspheres for advanced applications.     

Array biosensor for detection of toxins

The array biosensor is capable of detecting multiple targets rapidly and simultaneously on the surface of a single waveguide. Sandwich and competitive fluoroimmunoassays have been developed to detect high and low molecular weight toxins, respectively, in complex samples. Recognition molecules (usually antibodies) were first immobilized in specific locations on the waveguide and the resultant patterned array was used to interrogate up to 12 different samples for the presence of multiple different analytes. Upon binding of a fluorescent analyte or fluorescent immunocomplex, the pattern of fluorescent spots was detected using a CCD camera. Automated image analysis was used to determine a mean fluorescence value for each assay spot and to subtract the local background signal. The location of the spot and its mean fluorescence value were used to determine the toxin identity and concentration. Toxins were measured in clinical fluids, environmental samples and foods, with minimal sample preparation. Results are shown for rapid analyses of staphylococcal enterotoxin B, ricin, cholera toxin, botulinum toxoids, trinitrotoluene, and the mycotoxin fumonisin. Toxins were detected at levels as low as 0.5 ng mL^–1.     

Nano‐Graphene Oxide Based Multichannel Sensor Arrays towards Sensing of Protein Mixtures

Optical array‐based sensors are attractive candidates for the detection of various bio‐analytes due to their convenient fabrication and measurements. For array‐based sensors, multichannel arrays are more advantageous and used frequently in many electronic sensors. But most reported optically array based sensors are constructed on a single channel array. This difficulty is mainly instigated from the overlap in optical responses. In this report we have used nano‐graphene oxide (nGO) and suitable fluorophores as sensor elements to construct a multichannel sensor array for the detection of protein analytes. By using the optimized multichannel array we are able to detect different proteins and mixtures of proteins with 100 % classification accuracy at sub‐nanomolar concentration. This modified method expedites the sensing analysis as well as minimizes the use of both analyte and sensor elements in array‐based protein sensing. We have also used this system for the single channel array‐based sensing to compare the sensitivity and the efficacy of these two systems for other applications. This work demonstrated an intrinsic trade‐off associated with these two methods which may be necessary to balance for array‐based analyte detections.     

A single cataluminescence sensor based on spectral array and its use in the identification of vinegars

The discrimination of complex mixtures, especially those with very similar compositions, remains a challenging part of chemical analysis. In this paper, a single cataluminescence (CTL) sensor constructed using MgO nanomaterials in a closed reaction cell (CRC) was used to identify vinegars. It may provide an archetype of this type of highly multicomponent system. By scanning the CTL spectra, which were distributed in 15 wavelengths during the reaction period, the spectral array patterns of the vinegars were obtained. These functioned as their fingerprints. The CTL signals of the array were then normalized and identified through linear discrimination analysis (LDA). Nine types and eight brands of vinegars and an additional series of artificial samples were tested; the new technique was found to distinguish between them very well. This single sensor demonstrated excellent promise for analysis of complex mixtures in real-world applications and may provide a novel method for identifying very similar complex analytes.     

An integrated digital microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids

Clinical diagnostics is one of the most promising applications for microfluidic lab-on-a-chip systems, especially in a point-of-care setting. Conventional microfluidic devices are usually based on continuous-flow in microchannels, and offer little flexibility in terms of reconfigurability and scalability. Handling of real physiological samples has also been a major challenge in these devices. We present an alternative paradigm--a fully integrated and reconfigurable droplet-based "digital" microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids. The microdroplets, which act as solution-phase reaction chambers, are manipulated using the electrowetting effect. Reliable and repeatable high-speed transport of microdroplets of human whole blood, serum, plasma, urine, saliva, sweat and tear, is demonstrated to establish the basic compatibility of these physiological fluids with the electrowetting platform. We further performed a colorimetric enzymatic glucose assay on serum, plasma, urine, and saliva, to show the feasibility of performing bioassays on real samples in our system. The concentrations obtained compare well with those obtained using a reference method, except for urine, where there is a significant difference due to interference by uric acid. A lab-on-a-chip architecture, integrating previously developed digital microfluidic components, is proposed for integrated and automated analysis of multiple analytes on a monolithic device. The lab-on-a-chip integrates sample injection, on-chip reservoirs, droplet formation structures, fluidic pathways, mixing areas and optical detection sites, on the same substrate. The pipelined operation of two glucose assays is shown on a prototype digital microfluidic lab-on-chip, as a proof-of-concept.     

pH-sensitive polyelectrolyte complex gel microspheres composed of chitosan/sodium tripolyphosphate/dextran sulfate: swelling kinetics and drug delivery properties

Porous chitosan (CS) polyelectrolyte complex (PEC) hydrogel microspheres were prepared via either wet phase-inversion or ionotropic crosslinking with sodium tripolyphosphate (Na+ - TPP) and dextran sulfate (DS). The resulting microspheres were characterized using scanning electron microscopy (SEM) and elemental analysis (EA). The controlled release behavior of ibuprofen (IBU) from these microspheres was investigated. The PEC microspheres were about 700-950 microm in diameter with large pores and open porous structure. The CS/TPP/DS microspheres resisted hydrolysis in strong acid and biodegradation in enzymatic surroundings. The swelling kinetics for CS microspheres was close to Fickian diffusion, whereas those for CS/TPP and CS/TPP/DS were non-Fickian. Furthermore, the equilibrium water content (EWC) and water diffusion coefficient (D) increased with the pH of the media. The release profiles of IBU from CS/TPP/DS microspheres were slow in simulated gastric fluid (SGF, pH 1.4) over 3 h, but nearly all of the initial drug content was released in simulated intestinal fluid (SIF, pH 6.8) within 6 h after changing media. Overall the results demonstrated that CS/TPP/DS microspheres could successfully deliver a hydrophobic drug to the intestine without losing the drug in the stomach, and hence could be potential candidates as an orally administered drug delivery system.     

Thermodynamic enantioseparation behavior of phenylthiohydantoin‐amino acid derivatives in supercritical fluid chromatography on polysaccharide chiral stationary phases

Thirteen pairs of enantiomers belonging to the same structural family (phenylthiohydantoin‐amino acids) were analyzed on two polysaccharide chiral stationary phases, namely, tris‐(3,5‐dimethylphenylcarbamate) of amylose (Chiralpak AD‐H) or cellulose (Chiralcel OD‐H) in supercritical fluid chromatography with a carbon dioxide/methanol mobile phase (90:10 v/v). Five different temperatures (5, 10, 20, 30, 40°C) were applied to evaluate the thermodynamic behavior of these enantioseparations. On the cellulose stationary phase, the retention, and separation trends were most similar among the set of probe analytes, suggesting that the chiral cavities in this stationary phase have little diversity, or that all analytes accessed the same cavities. Conversely, the retention and separation trends on the amylose phase were much more diverse, and could be related to structural differences among the set of probe analytes (carbon chain length in the amino acid residue, secondary amine in proline, existence of covalent rings, or formation of pseudo‐rings via intramolecular hydrogen bonds). The large variability of behaviors on the amylose phase suggests that the chiral‐binding sites in this chiral stationary phase have more variety than on the cellulose phase, and that the analytes did access different cavities.     

Supramolecular chemistry approach to the design of a high-resolution sensor array for multianion detection in water

Reliable sensing of structurally similar anions in water is a difficult problem, and analytical tests and sensor devices for reliable sensing of multiple anions are very rare. This study describes a method for fabrication of simple colorimetric array-based assays for aqueous anion solutions, including complex analytes encountered in real-life applications. On the fundamental level, this method shows how the discriminatory capacity of sensor arrays utilizing pattern recognition operating in multianalyte environments may be dramatically improved by employing two key features. The synergy between the sensor and hydrogel host resembles the cooperative effects of an apoenzyme and cofactor: the host hydrogel helps extract the target anions from the bulk analyte while stripping the solvate molecules off the anions. In addition, the supramolecular studies of the affinity and selectivity of the potential sensors for target analytes allow for constructing an array predesigned for a particular analyte. To illustrate both aspects, an eight-sensor array utilizing colorimetric sensor materials showing selectivity for fluoride and pyrophosphate while displaying significant cross-reactivity for other anions such as carboxylates, phosphate, or chloride was used to differentiate between 10 anions. The quantitative analyses were also performed to show that the eight-sensor array was found to operate across 4 orders of magnitude concentrations (0.20-360 ppm; 10 microM to 20 mM). The applicability of this approach was demonstrated by analyzing several toothpaste brands. The toothpastes are complex analytes comprising both known and unknown anions in various concentrations. The fluoride-selective yet cross-reactive array is shown to utilize the fluoride content as the main differentiating factor while using the remaining anionic components for further differentiation between toothpaste brands.     

Individually addressable electrode array for multianalyte electrochemiluminescent immunoassay based on a sequential triggering strategy

Multianalyte immunoassay in a single run is often necessary to monitor or quantitate several components in a complex sample matrix for various purposes. In this paper we present a novel, individually addressable electrode array for sequential electrochemiluminescent (ECL) immunoassay using a non-array detector. An immunosensor array was fabricated by site-selectively immobilizing multiple antigens on different electrodes. With a competitive immunoassay format, the amounts of the bound Ru(bpy)(3)(2+) derivative labeled antibodies decreased with the increase of the antigens in the sample, and the ECL signals from different immunosensors were collected in turn by a photomultiplier with the aid of a home-made single-pore-three-throw switch. Using human IgG and rat IgG as model analytes, this multianalyte immunoassay showed detection limits down to 8.9 and 7.2 ng mL(-1) for them, respectively. The results for real sample analysis demonstrated that this strategy can provide a simple, sensitive, low-cost and high-throughput ECL immunosensor array for clinical diagnosis.     

Confinement effects on electromigration of long DNA molecules in an ordered cavity array

Confinement effects on the electromigration of long dsDNA molecules in an array of well-ordered, molecular sized cavities interconnected by nanopores are described. The array was prepared by replicating the structure of a colloidal crystal of microspheres in a polymer matrix. Both conformation and mobility studies show that the electrophoretic behavior of large DNA molecules is distinct from that in gels and other microfabricated sieves, showing a peak in mobility versus electric field, and an inversion in the separation order with field strength. A simple model was proposed to interpret this unexpected observation qualitatively. We conclude that the molecular behavior of DNA can be modified by the combination of entropic effect and electric trapping as a consequence of both unique geometry and conductivity of this cavity array material. This approach provides insights for design and optimization of on-chip molecular sieving structures for rapid separation of long DNA molecules. It may lead to a promising material for manipulation and size fractionation of other biological macromolecules.     

Selective sample treatment using molecularly imprinted polymers

The molecularly imprinted polymers (MIPs) are synthetic polymers possessing specific cavities designed for a target molecule. By a mechanism of molecular recognition, the MIPs are used as selective sorbents for the solid-phase extraction of target analytes from complex matrices. MIPs are often called synthetic antibodies in comparison with immuno-based sorbents; they offer some advantages including easy, cheap and rapid preparation and high thermal and chemical stability. This review describes the use of MIPs in solid-phase extraction with emphasis on their synthesis, the various parameters affecting the selectivity of the extraction, their potential to selectively extract analytes from complex aqueous samples or organic extracts, their on-line coupling with LC and their potential in miniaturized devices.     

Analysis of inflammatory biomarkers from tissue biopsies by chip-based immunoaffinity CE

To aid in the biochemical analysis of human skin biopsies, a semiautomatic chip-based CE system has been developed for measuring inflammatory biomarkers in microdissected areas of the biopsy. Following solubilization of the dissected tissue, the desired biomarkers were isolated by immunoaffinity capture using a panel of 12 antibodies, immobilized on a disposable glass fiber disk, within the extraction port of the chip. The captured analytes were labeled with a 635 nm light-emitting laser dye and electroeluted into the separation channel. Electrophoretic separation of all of the analytes was achieved in 2.2 min with quantification of each peak being performed by online LIF detection and integration of each peak area. Comparison of the results obtained from the chip-based system to those obtained using commercially available high-sensitivity immunoassays demonstrated that the chip-based assay provides a fast, accurate procedure for studying the concentrations of inflammatory biomarkers in complex biological materials. The degree of accuracy and precision achieved by the chip-based CE is comparable to conventional immunoassays and the system is capable of analyzing circa six samples per hour. With the ever-expanding array of antibodies that are commercially available, this chip-based system can be applied to a wide variety of different biomedical analyses.     

Differential receptor arrays and assays for solution-based molecular recognition

Nature has inspired an emergent supramolecular field of synthetic receptor arrays and assays for the pattern-based recognition of various bioanalytes and metal species. The synthetic receptors are not necessarily selective for a particular analyte, but the combined signal response from the array is diagnostic for the analyte. This tutorial review describes recent work in the literature for this emerging supramolecular field and details basic array and assay design principles. We review the analytes targeted, signaling types used, and pattern recognition.Developing specific receptors for the solution-based analysis of complex analytes and mixtures is a daunting task. A solution to this difficult task has been inspired by nature's use of arrays of receptors in the senses of taste and smell. An emerging field within supramolecular chemistry is the use of synthetic and readily available receptors in array formats for the detection of analytes in solution. Each receptor in a differential array does not necessarily have selectivity for a particular analyte, but the combined fingerprint response can be extracted as a diagnostic pattern visually, or using chemometric tools. This new genre of molecular recognition is advancing rapidly with several groups developing novel array platforms and receptors.     

A pneumatic micromixer facilitating fluid mixing at a wide range flow rate for the preparation of quantum dots

A novel fluid micromixer based on pneumatic perturbation and passive structures was developed. This micromixer facilitates integration and is applicable to fluid mixing over a wide range of flow rates. The microfluidic mixing device consists of an S-shaped structure with two mixing chambers and two barriers, and two pneumatic chambers designed over the S-shaped channel. The performance of the micromixer for fluids with wide variation of flow rates was significantly improved owing to the integration of the pneumatic mixing components with the passive mixing structures. The mixing mechanism of the passive mixing structures was explored by numerical simulation, and the influencing factors on the mixing efficiency were investigated. The results showed that when using a gas pressure of 0.26 MPa and a 100 m-thick polydimethylsiloxane (PDMS) pneumatic diaphragm, the mixing of fluids with flow rates ranging from 1 to 650 L/min was achieved with a pumping frequency of 50 Hz. Fast synthesis of CdS quantum dots was realized using this device. Smaller particles were obtained, and the size distribution was greatly improved compared with those obtained using conventional methods.     

Determination of eight illegal drugs in human urine by combination of magnetic solid‐phase extraction with capillary zone electrophoresis

Using magnetite/silica/poly(methacrylic acid‐co‐ethylene glycol dimethacrylate) (Fe₃O₄/SiO₂/poly(MAA‐co‐EDMA)) magnetic microspheres, a rapid and high‐throughput magnetic solid‐phase extraction coupled with capillary zone electrophoresis (MSPE‐CZE) method was developed for the determination of illegal drugs (ketamine, amphetamines, opiates, and metabolites). The MSPE of target analytes could be completed within 2 min, and the eight target analytes could be baseline separated within 15 min by CZE with 30 mM phosphate buffer solution (PBS, pH 2.0) containing 15% v/v ACN as background electrolyte. Furthermore, hydrodynamic injection with field‐amplified sample stacking (FASS) was employed to enhance the sensitivity of this MSPE‐CZE method. Under such optimal conditions, the limits of detection for the eight target analytes ranged from 0.015 to 0.105 μg/mL. The application feasibility of MSPE‐CZE in illegal drugs monitoring was demonstrated by analyzing urine samples, and the recoveries of target drugs for the spiked sample ranging from 85.4 to 110.1%. The method reproducibility was tested by evaluating the intra‐ and interday precisions, and relative standard deviations of <10.3 and 12.4%, respectively, were obtained. To increase throughput of the analysis, a home‐made MSPE array that has potential application to the treatment of 96 samples simultaneously was used.     

Optical colorimetric sensor arrays for chemical and biological analysis

In recent years, the sensor array has attracted much attention in the field of complex system analysis on the basis of its good selectivity and easy operation. Many optical colorimetric sensor arrays are designed to analyze multi-target analytes due to the good sensitivity of optical signal. In this review, we introduce the targeting analytes, sensing mechanisms and data processing methods of the optical colorimetric sensor array based on optical probes (including organic molecular probes, polymer materials and nanomaterials). The research progress in the detection of metal ions, anions, toxic gases, organic compounds, biomolecules and living organisms (such as DNA, amino acids, proteins, microbes and cells) and actual sample mixtures are summarized here. The review illustrates the types, application advantages and development prospects of the optical colorimetric sensor array to help broad readers to understand the research progress in the application of chemical sensor array.     

Ultrasensitive detection of aliphatic nitro-organics based on “turn-on” fluorescent sensor array

The broad class of explosives includes nitro aromatics as well as challenging aliphatic nitro-organics whose detection is important from counter-terrorism and national security perspectives. Here we report a turn-on fluorescent sensor array based on aggregation-induced emission (AIE) fluorophores as receptors. To achieve a good sensing system with fast response, good sensitivity and low detection limit, three receptors with abundant chemical diversities for target analytes were synthesized. The turn-on response of the individual receptor showed highly variable and cross-reactive analyte-dependent changes in fluorescence. The excellent ability to identify a variety of explosives, especially the challenging aliphatic nitro-organics (2,3-dimethyl-2,3-dinitrobutane (DMNB), 1,3,5-trinitro-1,3,5-triazinane (RDX), cyclotetramethylene tetranitramine (HMX) and entaerythritol tetranitrate (PETN)), was demonstrated in qualitative and quantitative analyses with 100% accuracy. The fluorescence signal amplification in the presence of explosives allows for application of these receptors in a sensor microarray suitable for high-throughput screening. These results suggested that the cross-reactive sensor array based on AIE fluorophores could find a wide range of applications for sensing various analytes or complex mixtures.