Christopher T. Culbertson

Microchip flow cytometry using electrokinetic focusing

Flow cytometry of fluorescently labeled and unlabeled latex particles is demonstrated on a microfabricated device. The latex particles were detected and counted using laser light scattering and fluorescence coincidence measurements. Sample confinement was accomplished using electrokinetic focusing at a cross intersection, and detection occurred 50 μm downstream from the intersection. Particles with diameters of 1 and 2 μm were analyzed and distinguished from each other based on their light scattering intensity and fluorescence. A maximum sample throughput of 34 particles/s was achieved. Sample mixtures with varying proportions of fluorescently labeled and unlabeled particles were also analyzed and found to be within experimental error of the expected ratios.

Micro total analysis systems: fundamental advances and biological applications.

It has been more than 20 years since the first micro total analysis systems (μTAS) papers were published. Initial reports of these devices, which are also commonly referred to as Labs-on-a-Chip (LOC), LabChips, microchips or microfluidic devices, generally focused on separations and the development of a variety of functional elements for sample manipulation and handling. One of the greatest potentials of μTAS, however, has always been in the integration of multiple functional elements to produce truly sample-in/answer-out systems. In the last decade, the march toward developing such integrated devices has accelerated significantly. Many μTAS reported now are quite sophisticated with multiple sample handling and processing steps that are highly integrated and often automated. While most of these devices are not yet strictly sample-in/answer-out several come quite close. There are, however, some significant hurdles still facing the development of true sample-in/answer-out systems especially in the areas of sample preparation, chip-to-real-world interfacing and detection. Additionally, further progress is needed in the miniaturization or elimination of external fluidic control elements. μTAS have found a major niche in the areas of biological and biomedical analyses, especially cellular and nucleic acid analysis. This focus on biological applications reflects the capabilities of these devices to precisely and accurately handle picoliter volumes of materials and to integrate cell transport, culturing or trapping with reagent delivery, and on-chip detection. Significant progress has been made in the development of a variety of cellular analysis systems; this field, however, is still rapidly growing and many papers focused on the expansion of such capabilities continue to be seen. Areas of focus remain the development of substrate materials and culturing conditions that do not unnaturally perturb or stress cells and that allow for extended culturing so that changes in cell physiology over time can be monitored. In addition, a significant amount of work has been directed to developing cell co-cultures on μTAS to mimic tissues, organs, and organ systems. μTAS can create unique, controlled environments to study cell-cell interactions that can not be replicated in any other way. For cellular assays substantial increases in throughput are also a focus. While significant development toward completely integrated cell assays has occurred and even some clinical demonstrations of such assays have been reported, the availability of commercial, fully integrated devices, however, has lagged. In addition to biological assays, the creative expansion of the basic μTAS toolkit with centrifugal platforms, digital microfluidics, and paper-based devices has substantially expanded its potential application base. Interest in these devices is generally more clinical in nature and again focused on generating sample-in/answer-out analyses. Significant work, however, is still needed for most of these platforms in terms of substrate materials, fluid control, sample handling, integration and throughput. Finally, the development of label-free detection technologies remains of interest. This review focuses on recent advances in μTAS technology in the areas of integrated biological assays and diagnostics with an analytical focus. We have also tried to highlight some material, fabrication, coating, separation, and detection advances with more general applicability. We have not included, for the most part, papers on synthesis, biosensors, theory, simulations or reviews. The papers included in this review were published between September 2012 and September 2013. The material was compiled using several strategies including extensive searches using Scifinder, Web of Science, PubMed, and Google Scholar. The contents of high impact journals were also scanned, including Analytical Chemistry, Lab-on-a-Chip, Nature, PNS, Appl. Phys., Letters, and Langmuir. Almost 2000 papers relating in some way to microfluidics were examined. We have done our best to try to identify some of the most interesting and promising papers and to report on them in this review. Without a doubt we have missed a few excellent papers and had to eliminate others based on space constraints and readability. For those papers that we have failed to include, we apologize in advance and welcome comments regarding any oversight that we have made.

Integrated microchip-device for the digestion, separation and postcolumn labeling of proteins and peptides

A microchip device was demonstrated that integrated enzymatic reactions, electrophoretic separation of the reactants from the products and post-separation labeling of proteins and peptides prior to detection. A tryptic digestion of oxidized insulin B-chain was performed in 15 min under stopped flow conditions in a heated channel, and the separation was completed in 1 min. Localized thermal control of the reaction channel was achieved using a resistive heating element. The separated reaction products were then labeled with naphthalene-2,3-dicarboxaldehyde (NDA) and detected by laser-induced fluorescence. A second reaction at elevated temperatures was also demonstrated for the on-chip reduction of disulfide bridges using insulin as a model protein. This device represents one of the highest levels, to date, of monolithic integration of chemical processes on a microchip.

High efficiency micellar electrokinetic chromatography of hydrophobic analytes on poly(dimethylsiloxane) microchips

This paper describes a simple method for the effective and rapid separation of hydrophobic molecules on polydimethylsiloxane (PDMS) microfluidic devices using Micellar Electrokinetic Chromatography (MEKC). For these separations the addition of sodium dodecyl sulfate (SDS) served two critical roles - it provided a dynamic coating on the channel wall surfaces and formed a pseudo-stationary chromatographic phase. The SDS coating generated an EOF of 7.1 x 10(-4) cm(2) V(-1) s(-1) (1.6% relative standard deviation (RSD), n = 5), and eliminated the absorption of Rhodamine B into the bulk PDMS. High efficiency separations of Rhodamine B, TAMRA (6-carboxytetramethylrhodamine, succinimidyl ester) labeled amino acids (AA), BODIPY FL CASE (N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester) labeled AAs, and AlexaFluor 488 labeled Escherichia coli bacterial homogenates on PDMS chips were performed using this method. Separations of Rhodamine B and TAMRA labeled AAs using 25 mM SDS, 20% acetonitrile, and 10 mM sodium tetraborate generated efficiencies > 100,000 plates (N) or 3.3 x 10(6) N m(-1) in <25 s with run-to-run migration time reproducibilities <1% RSD over 3 h. Microchips with 30 cm long serpentine separation channels were used to separate 17 BODIPY FL CASE labeled AAs yielding efficiencies of up to 837,000 plates or 3.0 x 10(6) N m(-1). Homogenates of E. coli yielded approximately 30 resolved peaks with separation efficiencies of up to 600,000 plates or 2.4 x 10(6) N m(-1) and run-to-run migration time reproducibilities of <1% RSD over 3 h.

Effects of the electric field distribution on microchip valving performance

Valving characteristics on microfluidic devices were controlled through manipulation of the electric field strengths during both the sample loading and dispensing steps. Three sample loading profiles for the constant volume valve (pinched injection) in conjunction with four dispensing schemes were investigated to study valving performance. The sample confinement profiles for the sample loading step consisted of a weakly pinched sample, a medium pinched sample, and a strongly pinched sample. Four dispensing schemes varied the electric field strengths in the sample and sample waste channels relative to the analysis channel to control the volume of the sample dispensed from the valve. The axial extent of the sample plug decreased as the electric field strengths in the sample and sample waste channels were raised relative to the analysis channel. In addition, a trade‐off existed between sample plug length and sensitivity.

Lowering the UV Absorbance Detection Limit in Capillary Zone Electrophoresis Using a Single Linear Photodiode Array Detector

A new approach for lowering the UV absorbance detection limit in capillary electrophoresis is presented. This approach involves the use of a photodiode array in which each of the diodes in the array is treated as an independent detector. Over the course of a run, therefore, an electropherogram is generated for each diode in the array. Averaging the electropherograms generated from 1500 diodes in a diode array resulted in a signal-to-noise ratio 85 times that of an electropherogram generated from any one diode in the array. These signal-to-noise improvements are discussed, and the detection limits are compared to the detection limits obtained from a commercial single-point detector. The array detector improves the detection limit by a factor of 3.8 (±0.4).

Micro Total Analysis Systems: Fundamental Advances and Applications

Applications Damith E. W. Patabadige,† Shu Jia,† Jay Sibbitts,† Jalal Sadeghi,†,‡ Kathleen Sellens,† and Christopher T. Culbertson*,† †Department of Chemistry, Kansas State University, 213 CBC Building, Manhattan, Kansas 66506, United States ‡Laser & Plasma Research Institute, Shahid Beheshti University, Evin, Tehran, 1983963113, Iran ■ CONTENTS Fundamentals 321 Fabrication 321 Materials and Bonding 321 3D Printing 322 Surface Modification 322 Functional Elements 323 Fluid Control 323 Acoustofluidics 323 Active Pumping 324 Valves 324 Gradient Formation 324 Mixing 324 Analyte Concentration 324 Separations 325 Detection 326 Electrochemical 326 Optical Detection 326 Surface Plasmon Resonance (SPR) 328 Surface Enhanced Raman Scattering (SERS) 328 Mass Spectrometry 328 Impedance 329 Conductivity 329 Other 329 Microfluidic Platforms 329 Integrated Devices 329 Digital Microfluidics 330 Point of Care (POC) 331 Centrifugal 331 World-to-Chip Interface 332 Applications 332 Drug Screening and Drug Discovery 332 Disease Diagnosis 332 Nucleic Acid Analysis 333 Hybridization Microarrays 333 Microbiomes and Environmental Changes 334 Chemotaxis 334 Extreme Environments 334 Protein Analysis 334 Conclusions and Outlooks 334 Author Information 335 Corresponding Author 335 Author Contributions 335 Notes 335 Biographies 335 Acknowledgments 335 References 335 I has been 14 years since the inaugural microfluidics review in Analytical Chemistry was published. The first papers describing these devices which are also commonly referred to as Micro Total Analysis Systems, Lab-on-a-Chip (LOC), LabChips, microchips, or microfluidic devices generally focused on separations and the development of a variety of fluidic structures for sample manipulation and handling. The ultimate premise behind the development of these devices, however, has always been their potential to perform complete, automated chemical analyses as many of its monikers imply. Since the first review there has been remarkable progress in terms of developing truly sample-in/answer-out microfluidic devices that integrate multiple functional elements. Reports of novel individual components for these devices have now been surpassed by reports of devices with integrated functional elements that can actually perform partially automated or fully automated chemical analyses. There are, however, still some significant challenges facing the development of microfluidic systems especially in the areas of sample preparation, chip-toreal-world interfacing, and detection. In terms of applications microfluidic devices have excelled in addressing a variety of biomedical analyses, especially those dealing with individual cells. This area has become so popular that it is now the subject of two additional Analytical Chemistry reviews, one on cellular analysis and the other on droplet microfluidics. There are, however, other application areas where the use microfluidic systems are popular, including drug screening, disease diagnosis, and nucleic acid analysis. In addition, the interest in developing clinical assays on paper microfluidic devices for use in resource poor situations has soared. This review focuses on recent advances in microfluidic technology with an analytical focus in the areas of fundamental advances, integrated devices, and biomolecular assays since the last review published in 2014. Reviews more tightly focused on cellular analysis and droplet microfluidics can be found elsewhere in this issue. We have tried to highlight some material, fabrication, coating, separation, and detection advances with more general applicability. We have not included, for the most part, papers on synthesis, biosensors, theory, simulations, or reviews. The papers included in this review were published between September 2013 and September 2015. The material was compiled using several strategies including

Manipulation of bacteriophages with dielectrophoresis on carbon nanofiber nanoelectrode arrays

This work describes efficient manipulation of bacteriophage virus particles using a nanostructured DEP device. The nonuniform electric field for DEP is created by utilizing a nanoelectrode array (NEA) made of vertically aligned carbon nanofibers versus a macroscopic indium tin oxide electrode in a “points‐and‐lid” configuration integrated in a microfluidic channel. The capture of the virus particles has been systematically investigated versus the flow velocity, sinusoidal AC frequency, peak‐to‐peak voltage, and virus concentration. The DEP capture at all conditions is reversible and the captured virus particles are released immediately when the voltage is turned off. At the low virus concentration (8.9 × 104 pfu/mL), the DEP capture efficiency up to 60% can be obtained. The virus particles are individually captured at isolated nanoelectrode tips and accumulate linearly with time. Due to the comparable size, it is more effective to capture virus particles than larger bacterial cells with such NEA‐basedDEP devices. This technique can be potentially utilized as a fast sample preparation module in a microfluidic chip to capture, separate, and concentrate viruses and other biological particles in small volumes of dilute solutions in a portable detection system for field applications.

Electrokinetic trapping using titania nanoporous membranes fabricated using sol-gel chemistry on microfluidic devices.

We have developed a new method for analyte preconcentration on a microfluidic device using a porous membrane fabricated via sol–gel chemistry. These porous membranes were fabricated within the channels of glass microfluidic devices exploiting laminar flow to bring an alcoholic sol–gel precursor (titanium isopropoxide in 2‐propanol) into contact with an alcohol–water solution at a channel cross intersection. These two streams reacted at the fluidic interface to form a porous titania membrane. The thickness of the membrane could be altered by changing the [H2O]. Analyte concentration was accomplished by applying a voltage across the titania membrane. The level of analyte enrichment was monitored, and enrichment factors of above 4000 in 400 s were obtained for 2,7‐dichlorofluorescein.

Monitoring intracellular nitric oxide production using microchip electrophoresis and laser-induced fluorescence detection

Nitric oxide (NO) is a biologically important short-lived reactive species that has been shown to be involved in a large number of physiological processes. The production of NO is substantially increased in immune and other cell types through the upregulation of inducible nitric oxide synthase (iNOS) caused by exposure to stimulating agents such as lipopolysaccharide (LPS). NO production in cells is most frequently measured via fluorescence microscopy using diaminofluorescein-based probes. Capillary electrophoresis with laser-induced fluorescence detection has been used previously to separate and quantitate the fluorescence derivatives of NO from potential interferences in single neurons. In this paper, microchip electrophoresis (ME) coupled to laser-induced fluorescence (LIF) detection is evaluated as a method for measurement of the NO production by Jurkat cells under control and stimulating conditions. ME is ideal for such analyses due to its fast and efficient separations, low volume requirements, and ultimate compatibility with single cell chemical cytometry systems. In these studies, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA) was employed for the detection of NO, and 6-carboxyfluorescein diacetate (6-CFDA) was employed as an internal standard. Jurkat cells were stimulated using lipopolysaccharide (LPS) to produce NO, and bulk cell analysis was accomplished using ME-LIF. Stimulated cells exhibited an approximately 2.5-fold increase in intracellular NO production compared to the native cells. A NO standard prepared using diethylamine NONOate (DEA/NO) salt was used to construct a calibration curve for quantitation of NO in cell lysate. Using this calibration curve, the average intracellular NO concentrations for LPS-stimulated and native Jurkat cells were calculated to be 1.5 mM and 0.6 mM, respectively