Surface-Enhanced Raman Spectroscopy of Analytes in Blood

Campos, Antonio Renteria, II University of Minnesota 2015 해외박사(DDOD)

Although Raman scattering has traditionally been considered a weak process, making analysis of low concentration analytes in complex matrices difficult, both methodological and instrumentation advances in the last couple decades have made Raman spectroscopy a viable and useful analytical tool. This is especially true for analyte species within aqueous environments because the Raman scattering cross-section of water is small; one particular example of a critical aqueous environment is analysis of and in blood. The work detailed in Chapter 1 will analyze much of the literature related to Raman analysis in blood within the last 20 years, including normal Raman, surface-enhanced Raman, and spatially offset Raman analyses. The first section will focus on direct analysis of blood samples, including determining the age of deposited or donated blood and blood content within body fluid mixtures. The second section will discuss intrinsic Raman-based detection of small molecules and protein analytes within blood as well as extrinsic Raman detection of tumors. The last section will review the recent use of spatially offset Raman and surface-enhanced spatially offset Raman spectroscopy to analyze molecular analytes, tissue, bone, tumors, and calcifications, including in vivo analysis. This focal point closes with perspective on critical gaps and upcoming developments for Raman analysis in blood. Raman detection in blood can be applied to different forensic fields and can also be used for the detection of foreign analytes. In current events, ricin has been discussed frequently because of letters sent to high-ranking government officials containing the easily extracted protein native to castor beans. Ricin B chain, commercially available and not dangerous when separated from the A chain, enables development of ricin sensors while minimizing the hazards of working with a bioterror agent that does not have a known antidote. As the risk of ricin exposure, common for soldiers, becomes increasingly common for civilians, there is a need for a rapid, real-time detection of ricin. To this end, aptamers have been used recently as an affinity agent to enable the detection of ricin in food products via surface-enhanced Raman spectroscopy (SERS) on colloidal substrates. One goal of this work is to extend ricin sensing into whole human blood; this goal required application of a commonly used plasmonic surface, the silver film-over-nanosphere (AgFON) substrate, which offers SERS enhancement factors of 106 in whole human blood for up to 10 days. This aptamer-conjugated AgFON platform enabled ricin B chain detection for up to 10 days in whole human blood. Principle component analysis (PCA) of the SERS data clearly identifies the presence or absence of physiologically relevant concentrations of ricin B chain in blood. In addition to the detection of ricin B chain at a relevant concentration, the development of a platform to perform a single experiment calibration curve was performed through the combination of microfluidic devices with SERS substrates. Microfluidic sensing platforms facilitate parallel, low sample volume detection using various optical signal transduction mechanisms. Herein, we introduce a simple mixing microfluidic device, enabling serial dilution of introduced analyte solution that terminates in five discrete sensing elements. We demonstrate the utility of this device with on-chip fluorescence and surface-enhanced Raman scattering (SERS) detection of analytes, and we demonstrate device use both when combined with a traditional inflexible SERS substrate and with SERS-active nanoparticles that are directly incorporated into microfluidic channels to create a flexible SERS platform. The results indicate, with varying sensitivities, that either flexible or inflexible devices can be easily used to create a calibration curve and perform a limit of detection study with a single experiment. In Chapter 4, the synthesis of an ultrastable and reversible pH nanosensor using gold nanosphere aggregates functionalized with 4-mercaptobenzoic acid (MBA) that are encapsulated in mesoporous silica was performed. The pH nanosensor is stable and functional in human whole blood for a period of more than 3 months. With the growing interest in nanoparticles and nanomaterials, a demonstration was organized for a high school AP Chemistry class. Spectrophotometry and colorimetry experiments are common in high school and college chemistry courses. Previous work has demonstrated that handheld camera devices can be used to quantify the concentration of a colored analyte in solution in place of traditional spectrophotometric or colorimetric equipment. Chapter 5 extends this approach to an investigation of a mesogold mineral supplement. With the addition of free Google applications, the investigation provides a feasible, sophisticated lab experience, especially for teachers with limited budgets.

Transport-kinetic processes and surface chemistry in biosensor design

Vijayendran, Ravi Albert University of Illinois at Urbana-Champaign 2001 해외박사(DDOD)

Biosensors are analytical devices that detect a target analyte on the basis of biomolecular recognition. Detection occurs as the consequence of specific interactions between the analyte and complementary biomolecules immobilized on the transducer surface. Several physicochemical factors influence this detection process. This thesis examines the role of these factors in sensor operation and also evaluates specific methods to manipulate these factors and improve sensor performance. We begin by investigating the kinetic and transport processes that underlie analyte recognition. A transport-kinetic model is developed to quantitatively relate these processes to sensor response in a typical biosensor measurement. Predictions from our model are compared with kinetic data from a fiber optic immunosensor. With these experimental comparisons, we demonstrate that our model provides a more physically rigorous description of analyte transport, and is thus better for data analysis and sensor design than competing models. The role of surface effects in biosensor operation is also addressed. We examine how immobilization impacts the activity of the biomolecules on the transducer surface. Although these molecules display homogeneous binding characteristics in solution, they often exhibit heterogeneous binding properties after surface immobilization. We measure binding isotherms and the detection kinetics for several analyte-receptor systems constructed with various immobilization strategies. By comparing theoretical models with experimental data, we elucidate the relationship between protein immobilization chemistry and receptor heterogeneity, and identify methods for constructing more uniformly reactive protein films. Finally, sample mixing is examined as a potential method to improve the performance of microfluidic biosensors. We attempt to mix sections of the sample solution where the analyte concentration is high with other sections where it is low, and thereby reduce the sensor response time when the detection kinetics are diffusion-limited. A serpentine micromixer, originally designed to mix two fluids in bulk solution via “chaotic advection,” is used to mix the sample as it passes through a surface plasmon resonance biosensor. These experiments indicate that such “solution-based” mixing strategies can be effective in microfluidic biosensors.

Applications and design modifications for portable mass spectrometers

Sanders, Nathaniel Louis Purdue University 2009 해외박사(DDOD)

The need for in situ chemical analysis in fields such as environmental or security analysis has lead to the development of detection methodologies using lab scale instrumentation. Because of the excellent analytical capabilities of mass spectrometry in terms of speed, sensitivity, specificity, and versatility, this technique has been investigated for achieving needed solutions. The solutions developed using traditional instruments have proven useful on a limited scale due to large instrument size and limitations in sample introduction. The purpose of this thesis is to demonstrate the analytical capabilities of multiple versions of home-built mass spectrometers by analysis of environmentally relevant analytes. The instruments investigated are characterized not only by their portability but by their use of atmospheric pressure inlets, allowing the introduction of externally generated ions. The instruments used are fully functional as indicated by mass ranges up to m/z 450, unit (or better) resolution, tandem mass spectrometric capabilities, and limits of detection in the low to sub-ppb range. The instruments also demonstrated the capability for further improvements in analytical parameters and versatility by physical and electronic modifications to the instrument. Ambient ionization methods developed in lab, such as desorption electrospray ionization (DESI) and low temperature plasma (LTP) ionization have allowed rapid, direct analysis of analyte from an untreated surface. Many highly relevant applications have been developed using these ionization methods on lab scale and handheld instruments. Modification of a handheld instrument has allowed the investigation of security screening experiments, namely the detection of explosives on a large area using ambient ionization methods.

On-chip isotachophoresis assays for high sensitivity electrophoretic preconcentration, separation, and indirect detection

Khurana, Tarun K Stanford University 2009 해외박사(DDOD)

The origins of microfluidics field lie in microanalytical methods such as gas chromatography, high pressure liquid chromatography and capillary electrophoresis that are revolutionizing the field of chemical analysis. Microfluidic devices have been particularly attractive for separation based chemical and biological analysis since the small length scales bring fundamental improvements in reagent volume use, analysis time, resolution, and separation efficiency. However, smaller length scales and volumes are also associated with lower detection sensitivity and therefore, this limits many applications to fluorescent analytes. This dissertation focuses on electrokinetic preconcentration and separation methods to improve the detection sensitivity on microchip platform and to extend the scope of microfluidic analysis to non-fluorescent analytes. Isotachophoresis (ITP) is a robust sample preconcentration technique that leverages spatial mobility gradients to focus analytes into zones that are ∼10 mum wide. Such extreme focusing of analytes results in drastic improvement in the detection sensitivity and resolution of electrophoretic separation system. We present a theoretical and experimental study of dynamics of ITP preconcentration that helps to identify optimum operating parameters to achieve high sample preconcentration. The theoretical study involves development of analytical models to identify the fundamental parameters governing the focusing dynamics and development of perturbation model and dispersion model to reduce the complexity of this numerically stiff problem. We performed controlled experiments to isolate the effect of each parameter influencing the preconcentration dynamics from others. These experimental results are used for validation of theoretical models and also serve as guidelines for ITP preconcentration assay design. We have also developed an indirect detection technique to detect non fluorescent analytes on standard microfluidic setup equipped with fluorescence detection platforms. We leverage ITP to preconcentrate and separate analytes into distinct analyte zones arranged in order of reducing electrophoretic mobility. Using a ladder of fluorescent species with different electrophoretic mobilities (termed as fluorescent mobility markers) to demarcate the boundaries of these analyte zones, we indirectly detect the non-fluorescent analytes present. We obtain ∼1 muM detection sensitivity with this assay with high repeatability and have demonstrated indirect detection of a variety of analytes such as amino acids, organic acids and environmental toxins such as phenols and cresol. Lastly, we demonstrate simultaneous electrophoretic preconcentration and separation of analytes in a single step injection process in off-the-shelf, standard microfluidic chips using isotachophoresis (ITP). Our technique leverages an electric field gradient between the leading and trailing electrolytes to concentrate analytes into distinct non-dispersing bands. This is the first experimental study to identify that the gradient results from slow reaction kinetics of hydration and carbamation of dissolved atmospheric carbon dioxide. We use a fluorescent counterion tracer technique to study the evolution of carbonate and carbamate zones and the electric field gradient region between them. Using this assay, we have demonstrated one step focusing and separation of 25 bp DNA ladder, and the fractionation of DNA and proteins. The technique has potential to simplify and improve the detection sensitivity of many microchip electrophoresis assays.

Colorimetric sensor arrays for the detection of aqueous and gaseous analytes

Musto, Christopher Joseph University of Illinois at Urbana-Champaign 2010 해외박사(DDOD)

The past decade has seen great interest concerning the development of artificial sensing devices; most notably optoelectronic tongues and noses. Utilizing previous research on how the mammalian gustatory and olfactory systems operate, significant progress in mimicking these systems has been realized. The turning point in this field of research has been the discovery that the mammalian senses of smell and taste are not based on specific receptors for each stimulant, but rather an array of semi-specific receptors that function simultaneously to produce a pattern. This pattern is interpreted in the brain, and classified either as a known stimulant or a new analyte similar to a known family of tastes or odors. As a predominantly visual species, we are programmed to acknowledge visible reports to chemical reactions over alternative reporting methods. Thus, colorimetric sensing can be more advantageous than other techniques and can allow for a greater number of chemical reactions to be probed. One colorimetric approach to sensing involves the immobilization of cross-responsive chemosensors capable of showing a color change upon reaction with analytes or mixtures of analytes. The employment of porous glasses as an immobilization technique has allowed for facile detection of analytes, both aqueous and gaseous, by allowing dye-analyte interactions to occur while preventing the sensor dye from escaping from the matrix. In this manner, colorimetric sensor arrays have been fashioned that are capable of discriminating among structurally similar compounds such as sugars, while retaining the ability to detect a wide range of analytes including toxic industrial chemicals. For aqueous detection, the newly developed porous glasses successfully immobilized otherwise soluble dyes that could detect changes in solution pH, caused by boronic aciddiol interactions. This allowed for rapid and sensitive detection and identification of natural and artificial sugars and sweeteners. Further experiments showed the array's ability to differentiate between a selection of common table-top sweeteners such as EqualRTM, Sweet'N'LowRTM, SplendaRTM, and natural sugars. Gas sensing applications were made possible by slight modifications to the liquid sensing array. Hydrophobic silica precursors were added to limit the effect of changing humidity on the array, and printing onto flat, non-porous polymer surfaces gave fast and easy accessibility of incoming analytes to the immobilized indicators. Stable and sensitive colorimetric arrays for the detection and semi-quantification of a large number of toxic industrial chemicals was made possible by the inclusion of additional indicators capable of colorimetrically reporting changes in polarity, metal ligation, and redox reactions. The performances of these sensing arrays showed extremely low limits of detection, and were capable of identifying toxic gases within a large range of concentrations; ppb up to concentration immediately dangerous to life and health. In order to improve upon the detection limits for weakly responding gaseous analytes, alternative methods were developed. It was found that the immobilization of simple and stable color-changing dyes within chemically-reactive matrices could allow for facile and sensitive detection and quantification of formaldehyde.

Application of polyatomic primary ions for organic secondary ion mass spectrometry

Diehnelt, Christopher Wayne Texas A&M University 2001 해외박사(DDOD)

Secondary ion mass spectrometry (SIMS) is a surface analytical technique capable of providing elemental, chemical, and spatial information about a surface. When equipped with a time-of-flight (TOF) mass analyzer, the surface sensitivity of the technique (TOF-SIMS) is maximized. The chief limitation for TOF-SIMS is the low yield of secondary ions produced per incident primary ion. Previous work has shown that the secondary ion yield can be increased through the application of polyatomic primary ions or through the use of designer substrates that enhance secondary ion emission. There are a large variety of polyatomic primary ions that can be used in TOF-SIMS, but there have been few attempts at accurate comparisons of primary ion performance. In this work, a detailed examination of the ancillary effects of polyatomic primary ion use and their influence on the effectiveness of polyatomic primary ions in TOF-SIMS was undertaken. Analyte specific secondary ion yields were measured from a variety of organic samples prepared as multilayer or monolayer surfaces. The amount of non-specific fragmentation, that is secondary ions that are ubiquitous, was observed to increase with the complexity of the primary ion. The amount of analyte-specific fragmentation, an indicator of the internal energy of the secondary ion population, increased with polyatomic primary ion bombardment and with sample thickness. The amount of metastable dissociation changed depending on primary ion type. The analyte specific secondary ion yield, the analyte specific fragmentation, and metastable fragmentation were combined to form an index that allowed comparison of multiple primary ions on the same sample. Results showed that polyatomic primary ion indeed improved the analysis of multilayer organic surfaces, when there is excess analyte, but for monolayer organic surfaces, atomic primary ions were most effective. A polyatomic primary ion was then used in conjunction with a coincidence counting protocol to examine the segregation and mixing within a mixed phospholipid bilayer. A model was developed which related the experimentally observed secondary ion correlations to the diameter of the segregation/domain.

Novel Analytical Methods for Improved Analysis of Biological Compounds

Beres, Martin Joseph The Ohio State University 2015 해외박사(DDOD)

The work contained within this dissertation focuses on innovative technologies in the field of analytical chemistry, particularly within high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Enhanced-fluidity liquids (EFL), which have low viscosity and high diffusivity, were studied as alternative mobile phases in mixed-mode hydrophilic interaction strong ion-exchange chromatography (HILIC/SCX). Additionally, these mobile phases were evaluated as environmentally friendly alternatives to traditional HILIC solvents in gradient separations. Finally, electrospun nanofibrous materials with high surface area to volume ratios were assessed as substrates in surface-assisted laser desorption/ionization mass spectrometry (SALDIMS). The potential of enhanced-fluidity liquid chromatography (EFLC) HILIC/SCX was explored, using amino acids as analytes. EFL mobile phases were prepared by adding liquefied CO2 to methanol:water (MeOH:H2O) mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The optimized chromatographic performance of these MeOH:H2O:CO 2 EFL mixtures was compared to traditional acetonitrile:water (ACN:H 2O) and MeOH:H2O liquid chromatography (LC) mobile phases. MeOH:H2O:CO2 mixtures offered higher efficiencies and resolution of the ten amino acids relative to the MeOH:H2O LC mobile phase, and decreased the required isocratic separation time by a factor of two relative to the ACN:H2O LC mobile phase. Large differences in selectivity were also observed between the EFLC and LC mobile phases. Retention mechanism studies revealed that the EFLC mobile phase separation was governed by a mixed-mode retention mechanism of HILIC/SCX. On the other hand, separations with ACN:H2O and MeOH:H2O LC mobile phases were strongly governed by only one retention mechanism, either HILIC or SCX, respectively. EFLC was then evaluated for "green" HILIC separations. The impact of CO2 addition to a MeOH:H2O mobile phase was studied as an alternative to traditional ACN:H2O HILIC mobile phases, while also optimizing buffer type, ionic strength, and pH. Using EFLC mixtures, a separation of 16 RNA nucleosides/nucleotides was achieved in 16 minutes with greater than 1.3 resolution for all analyte pairs. By using a reverse CO2 gradient, analysis time was reduced by over 100% in comparison to isocratic conditions. The optimal separation using MeOH:H2O:CO 2 mobile phases was also compared to that using MeOH:H2O and ACN:H2O mobile phases. Based on the chromatographic performance parameters (efficiency, resolution, and speed of analysis) and the overall environmental impact of the mobile phase mixtures, MeOH:H2O:CO 2 mixtures were preferred to ACN:H2O or MeOH:H2O mobile phases for the separation of mixtures of these RNA nucleosides and nucleotides. Finally, electrospun nanofibrous substrates were studied for the improvement of SALDI-MS analysis of large molecular weight proteins and polymers without the use of a chemical matrix. Various polymers (including polyacrylonitrile, polyvinyl alcohol, and SU-8 photoresist) and carbon substrates were examined. SALDI analysis using these substrates eliminated "sweet spot" formation typically seen in matrix-assisted laser desorption/ionization (MALDI), which lead to greater shot-to-shot reproducibility. The fiber diameter of these substrates played a significant role in the quality of the mass spectra generated, with smaller fiber diameter yielding higher signal to noise ratio (S/N). Additionally, the degree of pyrolysis also impacted the degree of fragmentation and overall S/N for the prepared carbon substrates.

Gas sensing mechanisms in chemiresistive metal phthalocyanine nanofilms

Bohrer, Forest I University of California, San Diego 2008 해외박사(DDOD)

Chemiresistive films of metallophthalocyanines (MPcs; M = Fe, Co, Ni, Cu, Zn, and H2) are shown to be sensitive to gas phase electron donors and acceptors. The mechanism of sensing occurs through coordination of the analyte molecule to metal center of the phthalocyanine; electron donors cause film current losses by trapping of charge carriers, while electron acceptors causes current gains by generation of charge carriers within the film. Vapor phase peroxides may cause gains or losses of film current by electrocatalytic processes dependent on the metal center. MPcs featuring varied metal centers and peripheral substituents are prepared via literature procedures. A novel route is devised for synthesis of a copper phthalocyanine incorporating the 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) group. MPc films are deposited by organic molecular beam epitaxy (OMBE) and spin-coating; film morphologies are examined by atomic force microscopy (AFM). It is demonstrated that substrate temperature during OMBE deposition can significantly alter grain morphology. Spin-coating offers a cost-effective alternative to OMBE, with soluble, functionalized phthalocyanines. The roles of solvent and functional group are explored and procedures for preparing uniform amorphous films are described. The differing mechanisms of sensing in metal-free phthalocyanine (H 2Pc) and metalated phthalocyanines (MPc) are examined with respect to electron-donating (basic) analytes. MPc sensitivities to vapor phase electron donors are correlated exponentially with analyte basicity as described by binding enthalpy, consistent with the van't Hoff equation and the standard free energy of reaction. Coordination of analytes to the phthalocyanine metal center (MPc) or inner protons (H2Pc) is the dominant mechanism of chemical sensing for basic analytes. Sensor recovery times t90 are demonstrated to depend exponentially on binding enthalpy. Linear discriminant analysis is used to identify analytes. Single sensor normalization of analyte concentration leads to excellent discrimination and identification of analytes. MPc sensing arrays are shown to be redox-selective vapor sensors of hydrogen peroxide and di-t-butyl peroxide. These peroxides cause unique current losses in CoPc sensors and current gains in FePc, NiPc, CuPc, ZnPc, and H2Pc sensors. Detection limits of 50 ppb and 250 ppb are achieved for hydrogen peroxide and di-t-butyl peroxide, respectively. Oxidation and reduction of peroxides via catalysis at the phthalocyanine surface is consistent with the pattern of sensor responses. Differential analysis by redox contrast of a small array of sensors thus uniquely identifies peroxide vapors. Chemically sensitive field-effect transistors (ChemFETs) of ZnPc are evaluated for use as vapor sensors. The average carrier mobility is 1.3x10 -4 cm2 V-1 s-1, comparable to previously reported phthalocyanine mobility values. ZnPc ChemFETs display persistent photoconductivity, lasting up to 1.5 months, which induces significant baseline drift. Persistent photoconductivity and sensor instability require improvements to the ZnPc ChemFET architecture before its implementation as vapor sensors.

SERS-based sensing platforms: Multilayer thin films, anisotropic nanoparticles, and core-shell structures

Mulvaney, Shawn Patrick The Pennsylvania State University 2001 해외박사(DDOD)

A technique capable of both qualitative and quantitative detection is Raman spectroscopy. Large enhancements in Raman scattering intensity can be realized when the analyte is in close proximity (<20 Å) to an appropriately roughened, noble metal surface. When analytes are examined in such a position the technique is known as surface enhanced Raman scattering (SERS), and enhancements as high as 10<super>14</super> have been reported. This thesis examines the improvement of SERS substrate performance. The second and third chapters describe the preparation and application of a multilayer metal film as a substrate for SERS. A novel substrate architecture was created by evaporating a discontinuous film of Ag over an Ag-clad colloidal Au submonolayer. This solid support substrate has an enhancement of 2 × 10<super> 6</super> and better than 15% reproducibility in signal. Analytes in more complex sample matrices can be examined when a thin film of polydimethyl siloxane (PDMS) is spin coated over the substrate. The PDMS film acts as a solid phase microextraction (SPME) medium, thereby concentrating hydrophobic analyte molecules near the SERS-active surface. Analytes positioned between two metal surfaces have enlarged Raman scatting because the signal is modulated by both metal surfaces. Chapter 4 addresses the deterministic preparation of these geometries, known as SERS sandwiches, that will be suitable for detailed study. Au-Ag-Au, rod-shaped nanoparticles can be prepared by membrane templated electrodeposition. Once these nanoparticles are immobilized on a surface the Ag strip can be etched leaving a closely space two particle surface feature. Details of preparing suitable films for the study of SERS sandwiches are reported. The final chapter describes the development of a SERS-active, core-shell particle to be used as a tagging system in bioassays. Glass-coated, analyte-tagged nanoparticles (GANs) are core-shell structures where a nanometer scale Au or Ag core is functionalized with a Raman active molecule and encapsulated in a glass shell. GANs particles are identified by the Raman spectrum of the attached Raman-tag. Scattering from that Raman-tag is amplified through SERS. Furthermore, biorecognition chemistry (i.e. nucleic acids, antibodies, antigens) can be attached to the glass shell without interfering with the Raman response.

Multiplexed detection methods for microchannel and conventional capillary electrophoresis

McReynolds, Jennifer Ann University of Illinois at Chicago 2005 해외박사(DDOD)

Improving detection techniques for capillary and microchannel electrophoresis is a key challenge. The difficulty stems from the narrow capillaries utilized for conventional CE and the reduced inner diameters of the separation channel in MCE. These small dimensions yield short optical pathlengths which create an obstacle in collecting signal from the separated analytes. Also, small, sometimes very dilute, sample volumes are being analyzed in these narrow capillaries creating an even greater detection challenge. The utilization of multiplex detection methods for CE and MCE separations to improve detection are described. The term multiplex is based on collecting signal from 'multiple detection windows' rather than a single window. Encoded detection techniques are multiplexed detection methods that can significantly improve detection. These encoded data is subsequently decoded post data collection for improving detection. Two types of encoded detection techniques, the Shah convolution Fourier transform (SCOFT) and the Hadamard transform (HT), are performed with a custom-built laser-induced fluorescence detection system with a multichannel detector. In SCOFT, the convolution detection is performed by sampling an analyte band migrating along the separation channel at its characteristic speed at evenly spaced regions. The sum of these sample peaks is the time-domain signal that can be Fourier transformed allowing the analyte to be viewed in terms of its characteristic velocity. Greater S/N enhancements and flexibility for this encoded detection format are illustrated. The HT detection technique is based on a weighing design principle of accurately weighing a number of objects in groups, rather than one at a time. After decoding, the mean square error can be significantly reduced and the S/N enhanced. Analytical signals from the separated analytes are collectively measured generating an enhancement in S/N after the decoding process. The extension of this multiplexed method towards absorbance detection in conventional CE for biological samples is also presented. The multiplex detection research described has provided significant detection enhancements for CE and MCE. This research has improved detection of less concentrated, small volume samples separated in the narrow channel geometry of the microchannels and capillaries. The multiplexed methods described in this research will aid in the improvement of separation analysis.