Photothermal Backscatter Interferometry for Enhanced Detection in Capillary Electrophoresis.

Refractive index (RI) detection using backscatter interferometry (BSI) enables universal detection in capillary electrophoresis (CE). BSI detection is a versatile on-capillary approach that is easily integrated with capillary or microfluidic channels, straightforward to miniaturize, and inexpensive. The focused BSI light source can also double as the excitation source for fluorescence, enabling simultaneous universal (BSI) and specific (fluorescence) signals from the same detection volume. To improve BSI detection and expand orthogonal content, we integrate photothermal absorption with BSI detection. Nonradiative relaxation of an excited analyte releases heat into the surroundings, which modifies both the local RI and conductivity (viscosity) of the analyte zone. We recently showed that the BSI signal is sensitive to both RI and conductivity, which makes photothermal absorption a promising route to signal enhancement. Here, we use coaxially delivered BSI and photothermal absorption beams to characterize BSI, photothermal BSI, and fluorescence detection using the separation of test samples. We show that photothermal absorption leads to 3 orders of magnitude improvement in BSI detection limits at the powers studied and provides new opportunities for studying binding interactions with CE.

Investigation of induced electroosmotic flow in small-scale capillary electrophoresis devices: Strategies for control and reversal.

Electroosmotic flow (EOF) is the bulk flow of solution in a capillary or microchannel induced by an applied electric potential. For capillary and microchip electrophoresis, the EOF enables analysis of both cations and anions in one separation and can be varied to modify separation speed and resolution. The EOF arises from an electrical double layer at the capillary wall and is normally controlled through the pH and ionic strength of the background buffer or with the use of additives. Understanding and controlling the electrical double layer is therefore critical for maintaining acceptable repeatability during method development. Surprisingly, in fused silica capillaries at low pH, studies observe an EOF even though the capillary surface should be neutralized. Previous work has suggested the presence of an "induced electroosmotic flow" from radial electric fields generated across the capillary wall due to the separation voltage and grounded components external to the capillary. Using thin-wall (15 µm) fused silica separation capillaries to facilitate the study of radial fields, we show that the EOF mobility depends on both the separation voltage and the location of external grounds. This is consistent with the induced EOF model, in which radial electric fields embed positive charges at the capillary walls to create an electrical double layer. The magnitude of the effect is characterized and shown to have long-range influences that are difficult to completely null by moving grounded components away from the separation capillary. Instead, active EOF control using externally applied potentials or a passive approach using a negative separation voltage are discussed as two possible methods for controlling the induced EOF. Both methods can reverse the EOF and improve the resolution and peak efficiency in amino acid separations.

Sample plug induced peak splitting in capillary electrophoresis studied using dual backscattered interferometry and fluorescence detection.

The appearance of unexpected peaks in capillary electrophoresis (CE) is common and can lengthen the time of method development as assay conditions and experimental parameters are varied to understand and mitigate the effects of the additional peaks. Additional peaks can arise when a single-analyte zone is split into multiple zones. Understanding the underlying mechanism of these phenomena, recognizing conditions that favor its presence, and knowing how to confirm and eliminate the effect are important for efficient method optimization. In this study, we examine how the overlap of analyte zones with the sample plug can lead to peak splitting. This is explored experimentally using dual detection CE, which enables both the sample plug and analyte zones to be independently and simultaneously measured from the same detection volume. Simulations performed via COMSOL Multiphysics confirm the origin of the splitting and help guide experiments to reduce and eliminate the effect. Our findings show that this peak splitting mechanism can arise in separations of both small and large molecules but is, especially, prevalent in separations of slowly migrating macromolecules. This effect is also more prevalent when using a short length-to-detector, as is commonly found in microfluidic applications. A simple diffusion-less model is introduced to develop strategies for reducing peak splitting that avoids modifying the apparatus, such as by lengthening the separation length, which can be difficult. Decreasing the sample plug length and slowing the electroosmotic flow can both reduce this effect, which is confirmed experimentally.

High-Speed Capillary Electrophoresis Using a Thin-Wall Fused-Silica Capillary Combined with Backscatter Interferometry.

High-speed capillary electrophoresis (HSCE) is implemented using a 10 cm total length fused-silica capillary (50 µm i.d., 80 µm o.d.) combined with refractive index (RI) detection using backscatter interferometry (BSI). The short capillary length reduces analysis time while the ultrathin wall (15 µm) efficiently dissipates heat from the separation channel, mitigating the deleterious effects of Joule heating. The separation capillary is mounted on a temperature-controlled heat sink that stabilizes the temperature to ±0.004 °C. This temperature stabilization improves separation efficiency and enhances RI detection. Ohm's Law plots confirm the superior heat dissipation of the HSCE capillary compared to a similarly prepared conventional CE capillary (50 µm i.d., 363 µm o.d.). The speed and efficiency of HSCE combined with universal RI detection is illustrated through the separation of K+, Ba2+, Mg2+, Na+, Li+, and Tris+ in approximately 30 s, with efficiencies greater than 500 000 plates/m. Run-to-run repeatability is explored using nine consecutive electrokinetic injections of a K+, Na+, and Li+ mixture. The average migration times and %RSD for K+, Na+, and Li+ were measured to be 22.04 s (1.59%), 26.81 s (1.38%), and 29.80 s (2.21%), respectively. Finally, we show that the BSI signal is sensitive to the separation voltage through the Kerr mechanism. This leads to peaks in the electropherogram from the injection process that are useful for precisely defining the start of each separation and quantifying the amount of sample injected onto the capillary.

Wavelength Modulated Back-Scatter Interferometry for Universal, On-Column Refractive Index Detection in Picoliter Volumes.

Wavelength-modulated back scatter interferometry (M-BSI) is shown to improve the detection metrics for refractive index (RI) sensing in microseparations. In M-BSI, the output of a tunable diode laser is focused into the detection zone of a separation channel as the excitation wavelength is rapidly modulated. This spatially modulates the observed interference pattern, which is measured in the backscattered direction. Phase-sensitive detection using a split photodiode detector aligned on one fringe of the interference pattern is used to monitor RI changes as analytes are separated. Using sucrose standards, we report a detection limit of 700 µg/L in a 75 µm i.d. capillary at the 3σ level, corresponding to a detection volume of 90 pL. To validate the approach for electrophoretic separations, Na+ and Li+ were separated and detected with M-BSI and indirect-UV absorbance on the same capillary. A 4 mg/L NaCl and LiCl mixture leads to comparable separation efficiencies in the two detection schemes, with better signal-to-noise in the M-BSI detection, but less baseline stability. The latter arises in part from Joule heating, which influences RI measurements through the thermo-optic properties of the run buffer. To reduce this effect, a 25 µm i.d. capillary combined with active temperature control was used to detect the separation of sucrose, glucose, and lactose with M-BSI. The lack of suitable UV chromophores makes these analytes challenging to detect directly in ultrasmall volumes. Using a 55 mM NaOH run buffer, M-BSI is shown to detect the separation of a mixture of 174 mg/L sucrose, 97 mg/L glucose, and 172 mg/L lactose in a 15 pL detection volume. The universal on-column detection in ultrasmall volumes adds new capabilities for microanalysis platforms, while potentially reducing the footprint and costs of these systems.

Scanning resonator microscopy integrating phase sensitive detection.

Scanning resonator microscopy (SRM) is a scanning probe technique that uses a small, optical resonator attached to the end of a conventional atomic force microscopy cantilever to simultaneously measure optical and topography properties of sample surfaces. In SRM, whispering gallery mode (WGM) resonances excited in the attached optical resonator shift in response to changes in surface refractive index (RI), providing a mechanism for mapping RI with high spatial resolution. In our initial report, the SRM tip was excited with a fixed excitation wavelength during sample scanning, which limits the approach. An improved method based on a wavelength modulation coupled with phase sensitive detection is reported here. This results in real-time characterization of WGM spectral shifts while eliminating complications arising from measurements based solely on signal intensity. This improved approach, combined with a modified tip design enabling integration of smaller resonators, is shown to enhance signal-to-noise and lead to sub-100 nm spatial resolution in the SRM optical image. The improved capabilities are demonstrated through measurements on thin dielectric and polymer films.

Integrating Whispering Gallery Mode Refractive Index Sensing with Capillary Electrophoresis Separations Using Phase Sensitive Detection.

Whispering gallery mode (WGM) resonators are small, radially symmetric dielectrics that recirculate light through continuous total internal reflection. High-Q resonances are observed that shift in response to changes in surrounding refractive index, leading to many applications in label-free sensing. Surface binding measurements with WGM resonators have demonstrated competitive analytical detection metrics compared to other sensing schemes. Similar figures of merit for detecting bulk refractive index changes, however, have proven more challenging. This has limited their use in applications such as capillary electrophoresis (CE), where their compact footprint and refractive index sensitivity offers advantages in nondestructive, universal detection. Here we couple WGM detection with CE by introducing a modulation scheme to improve detection limits. Phase sensitive WGM (PS-WGM) detection is developed to monitor real-time shifts in the WGM spectrum due to changes in surrounding refractive index. We directly compare phase sensitive detection with spectral measurements normally used to track WGM shifts. We report an improvement in detection limits by almost 300-fold using the PS-WGM method. The integrated CE with PS-WGM approach is demonstrated by detecting the separation of a three-component mixture of cations (Na(+), Li(+), and K(+)).

Interaction of cholesterol in ternary lipid mixtures investigated using single-molecule fluorescence.

Fluorescence measurements of the sterol analog 23-(dipyrrometheneboron difluoride)-24-norcholesterol (BODIPY-cholesterol) are used to compare the effects of cholesterol (Chol) in monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and chicken egg sphingomyelin (SM)/DOPC/Chol. Monolayers are formed using the Langmuir-Blodgett technique and compared at surface pressures of 8 and 30 mN/m. In particular, these ternary lipid mixtures are compared using both ensemble and single-molecule fluorescence measurements of BODIPY-cholesterol. In mixed monolayers incorporating 0.10 mol % BODIPY-cholesterol, fluorescence microscopy measurements as a function of cholesterol added reveal similar trends in monolayer phase structure for both DPPC/DOPC/Chol and SM/DOPC/Chol films. With a probe concentration reduced to ∼10(-8) mol % BODIPY-cholesterol, single-molecule fluorescence measurements using defocused polarized total internal reflection microscopy are used to characterize the orientations of BODIPY-cholesterol in the monolayers. Population histograms of the BODIPY emission dipole tilt angle away from the membrane normal reveal distinct insertion geometries with a preferred angle observed near 78°. The measured angles and populations are relatively insensitive to added cholesterol and changes in surface pressure for monolayers of SM/DOPC/Chol. For monolayers of DPPC/DOPC/Chol, however, the single-molecule measurements reveal significant changes in the BODIPY-cholesterol insertion geometry when the surface pressure is increased to 30 mN/m. These changes are discussed in terms of a squeeze-out mechanism for BODIPY-cholesterol in these monolayers and provide insight into the partitioning and arrangement of BODIPY-cholesterol in ternary lipid mixtures.

Electric field-enhanced backscatter interferometry detection for capillary electrophoresis.

Backscatter interferometry (BSI) is a refractive index (RI) detection method that is easily integrated with capillary electrophoresis (CE) and is capable of detecting species ranging from inorganic ions to proteins without additional labels or contrast agents. The BSI signal changes linearly with the square of the separation voltage which has been used to quantify sample injection, but has not been explored as a potential signal enhancement mechanism in CE. Here we develop a mathematical model that predicts a signal enhancement at high field strengths, where the BSI signal is dominated by the voltage dependent mechanism. This is confirmed in both simulation and experiment, which show that the analyte peak area grows linearly with separation voltage at high field strengths. This effect can be exploited by adjusting the background electrolyte (BGE) to increase the conductivity difference between the BGE and analyte zones, which is shown to improve BSI performance. We also show that this approach has utility in small bore capillaries where larger separation fields can be applied before excess Joule heating degrades the separation. Unlike other optical detection methods that generally degrade as the optical pathlength is reduced, the BSI signal-to-noise can improve in small bore capillaries as the larger separation fields enhance the signal.

Integration of microsphere resonators with bioassay fluidics for whispering gallery mode imaging.

Whispering gallery mode resonators are small, radially symmetric dielectrics that trap light through continuous total internal reflection. The resonant condition at which light is efficiently confined within the structure is linked with refractive index, which has led to the development of sensitive label-free sensing schemes based on whispering gallery mode resonators. One resonator design uses inexpensive high index glass microspheres that offer intrinsically superior optical characteristics, but have proven difficult to multiplex and integrate with the fluidics for sample delivery and fluid exchange necessary for assay development. Recently, we introduced a fluorescence imaging approach that enables large scale multiplexing with microsphere resonators, thus removing one obstacle for assay development. Here we report an approach for microsphere immobilization that overcomes limitations arising from their integration with fluidic delivery. The approach is an adaptation of a calcium-assisted glass bonding method originally developed for microfluidic glass chip fabrication. Microspheres bonded to glass using this technique are shown to be stable with respect to fluid flow and show no detectable loss in optical performance. Measured Q-factors, for example, remain unchanged following sphere bonding to the substrate. The stability of the immobilized resonators is further demonstrated by transferring lipid films onto the immobilized spheres using the Langmuir-Blodgett technique. Bilayers of DOPC doped with GM1 were transferred onto immobilized resonators to detect the binding of cholera toxin to GM1. Binding curves generated from shifts in the whispering gallery mode resonance result in a measured Kd of 1.5 × 10(-11) with a limit of detection of 3.3 pM. These results are discussed in terms of future assay development using microsphere resonators.

Single molecule probes of membrane structure: orientation of BODIPY probes in DPPC as a function of probe structure.

Single molecule fluorescence measurements have recently been used to probe the orientation of fluorescent lipid analogs doped into lipid films at trace levels. Using defocused polarized total internal reflection fluorescence microscopy (PTIRF-M), these studies have shown that fluorophore orientation responds to changes in membrane surface pressure and composition, providing a molecular level marker of membrane structure. Here we extend those studies by characterizing the single molecule orientations of six related BODIPY probes doped into monolayers of DPPC. Langmuir-Blodgett monolayers transferred at various surface pressures are used to compare the response from fluorescent lipid analogs in which the location of the BODIPY probe is varied along the length of the acyl chain. For each BODIPY probe location along the chain, comparisons are made between analogs containing phosphocholine and smaller fatty acid headgroups. Together these studies show a general propensity of the BODIPY analogs to insert into membranes with the BODIPY probe aligned along the acyl chains or looped back to interact with the headgroups. For all BODIPY probes studied, a bimodal orientation distribution is observed which is sensitive to surface pressure, with the population of BODIPY probes aligned along the acyl chains increasing with elevated surface pressure. Trends in the single molecule orientations for the six analogs reveal a configuration where optimal placement of the BODIPY probe within the acyl chain maximizes its sensitivity to the surrounding membrane structure. These results are discussed in terms of balancing the effects of headgroup association with acyl chain length in designing the optimal placement of the BODIPY probe.

Dual detection high-speed capillary electrophoresis for simultaneous serum protein analysis and immunoassays.

Serum protein electrophoresis (SPE) separates serum proteins into bands whose shape and amplitude can alert clinicians to a range of disorders. This is followed by more specific immunoassays to quantify important antigens and confirm a diagnosis. Here we develop a high-speed capillary electrophoresis (HSCE) platform capable of simultaneous SPE and immunoassay measurements. A single laser excitation source is focused into the detection zone of the capillary to measure both refractive index (SPE) and fluorescence signals (immunoassays). The refractive index signal measures characteristic SPE profiles for human serum separated in 100 mM boric acid (pH 10), 100 mM arginine (pH 11), and 20 mM CHES (pH 10). For the immunoassay, the fluorescence electropherograms reveal that CHES provides the optimal buffer for measuring the immunocomplex and separating it from the free antigen. Immunoassays in CHES yield a LOD of 23 nM and a LOQ of 70 nM for the detection of fluorescein. The high pH reduces protein adsorption but reduces antibody affinity. Preliminary studies carried out in 50 mM barbital at pH 8 show improved stability of the immunocomplex and better separation for immunoassay quantification. Further optimization will open new capabilities for measuring orthogonal diagnostic signals in seconds with HSCE.

Hydration effects on membrane structure probed by single molecule orientations.

Single molecule fluorescence measurements are used to probe the structural changes in glass-supported DPPC bilayers as a function of relative humidity (RH). Defocused polarized total internal reflection fluorescence microscopy is employed to determine the three-dimensional orientation of the fluorescent lipid analogue BODIPY-PC, doped into DPPC membranes in trace amounts. Supported DPPC bilayers formed using vesicle fusion and Langmuir-Blodgett/Langmuir-Schäfer (LB/LS) transfer are compared and show similar trends as a function of relative humidity. Population histograms of the emission dipole tilt angle reveal bimodal distributions as observed previously for BODIPY-PC in DPPC. These distributions are dominated by large populations of BODIPY-PC molecules with emission dipoles oriented parallel (≥81°) and normal (≤10°) to the membrane plane, with less than 25% oriented at intermediate tilts. As the relative humidity is increased from 13% to 95%, the population of molecules oriented normal to the surface decreases with a concomitant increase in those oriented parallel to the surface. The close agreement in trends observed for bilayers formed from vesicle fusion and LB/LS transfer supports the assignment of an equivalent surface pressure of 23 mN/m for bilayers formed from vesicle fusion. At each RH condition, a small population of BODIPY-PC dye molecules are laterally mobile in both bilayer preparations. This population exponentially increases with RH but never exceeds 6% of the total population. Interestingly, even under conditions where there is little lateral diffusion, fluctuations in the single molecule orientations can be observed which suggests there is appreciable freedom in the acyl chain region. Dynamic measurements of single molecule orientation changes, therefore, provide a new view into membrane properties at the single molecule level.

Direct detection of inorganic ions and underivatized amino acids in seconds using high-speed capillary electrophoresis coupled with back-scatter interferometry.

High speed capillary electrophoresis (HSCE) combined with refractive index (RI) detection is developed for the rapid separation and detection of inorganic ions and amino acids. A mixture of three inorganic ions (K+, Na+, Li+) and eight amino acids (Lys, Arg, Ala, Gly, Val, Thr, Trp, Asp) are detected using back scatter interferometry (BSI), without the need for chemical modifications or contrast. A thin-walled separation capillary (50 µm i.d. by 80 µm o.d.) helps mitigate Joule heating at the high field strengths required for rapid separations. This, combined with a short 8 cm length-to-detector (10 cm total length), enables separations on the seconds time scale. Using a background electrolyte (BGE) of 4 M acetic acid (pH 1.6) and a field strength of 900 V cm-1, all 11 analytes are separated in less than 40 s. Moreover, peaks in the BSI signal arising from the sample injection and EOF, enable electrophoretic mobilities to readily be obtained from apparent mobilities. This leads to excellent repeatability, with analyte electrophoretic mobilities varying from 0.39 to 1.56 % RSD over eight consecutive separations. The universal detection of inorganic ions and amino acids without prior chemical modification or additives in the BGE is an advantage of refractive index detection. A disadvantage arises from modest detection limits. Here, however, we show that submicromolar detection is possible with careful thermostatting of the thin separation capillary. A series of electropherograms are used to quantify arginine concentrations from 700 nM to 500 µM, using 50 µM Li+ as an internal standard. The resulting calibration curve leads to a calculated LOD of 376 nM and a LOQ of 1.76 µM. Diagnostically relevant amino acid panels are also separated, illustrating the potential for future applications in neurodegenerative and metabolic disease diagnostics. HSCE combined with BSI detection, therefore, is shown to be a rapid, sensitive, and universal approach for analyzing sample mixtures.

Near-field scanning optical microscopy: a tool for nanometric exploration of biological membranes.

Near-field scanning optical microscopy (NSOM) is an emerging optical technique that enables simultaneous high-resolution fluorescence and topography measurements. Here we discuss selected applications of NSOM to biological systems that help illustrate the utility of its high spatial resolution and simultaneous collection of both fluorescence and topography. For the biological sciences, these attributes seem particularly well suited for addressing ongoing issues in membrane organization, such as those regarding lipid rafts, and protein-protein interactions. Here we highlight a few NSOM measurements on model membranes, isolated biological membranes, and cultured cells that help illustrate some of these capabilities. We finish by highlighting nontraditional applications of NSOM that take advantage of the small probe to create nanometric sensors or new modes of imaging.

Exploring the effects of sterols in model lipid membranes using single-molecule orientations.

Single-molecule orientations of the fluorescent lipid analogue BODIPY-PC doped into lipid monolayers and bilayers of DPPC are used to characterize the structure present in the films as a function of sterol content. Out-of-focus polarized total internal reflection fluorescence microscopy (PTIRF-M) measurements are used to characterize the single-molecule tilt angles with respect to the membrane normal. Tilt angle histograms for Langmuir-Blodgett monolayers of DPPC reveal bimodal distributions at all surface pressures studied. A linear dependence in the dye population oriented normal to the membrane plane with surface pressure is found and used to characterize the equivalent surface pressure of supported bilayers formed through vesicle fusion. These measurements reveal an equivalent surface pressure of approximately 23 mN/m, which is somewhat lower than the currently accepted value of approximately 30-35 mN/m.(1-7) The effect of cholesterol, ergosterol, and lanosterol on membrane structure is also compared between DPPC bilayers and monolayers transferred at approximately 23 mN/m. The addition of cholesterol leads to dramatic changes in the tilt angle histograms while lanosterol has essentially no effect. The addition of ergosterol has a slight influence at higher concentrations. Using the average tilt angle calculated from the single-molecule histograms, the order parameter S is calculated as a function of cholesterol and compared with previous studies.(8-10).

Vault ribonucleoprotein particles and the central mass of the nuclear pore complex.

Nuclear pore complexes (NPCs) are macromolecular pores that span the nuclear envelope and undergo conformational changes in response to changes in cisternal calcium levels. Depletion of cisternal calcium leads to the appearance of a mass within the pore. The identity and role of this central mass remain unknown, although some studies suggest they are vault complexes. Vault complexes are 13 MDa ribonucleoproteins found in the cytoplasm and recently in the nuclei of some species, suggesting that they associate with NPCs to cross the nuclear envelope. Using Förster resonance energy transfer (FRET) measurements between labeled vaults and NPCs, we find significant energy transfer suggesting that vaults and NPCs are closely associated at the nuclear envelope. This is supported by high-resolution electron microscopy measurements revealing significant spatial correlations between gold-labeled vaults and NPCs. As the location of the central mass in the NPC is dependent on cisternal calcium levels, FRET signals under conditions of varying cisternal calcium were also measured and shown to undergo significant changes. Together, these findings suggest that the central mass observed in NPCs may be, at least in part, due to the presence of vaults in the pore. Possible roles in cyto-nuclear trafficking are discussed.

Characterization of power induced heating and damage in fiber optic probes for near-field scanning optical microscopy.

Tip-induced sample heating in near-field scanning optical microscopy (NSOM) is studied for fiber optic probes fabricated using the chemical etching technique. To characterize sample heating from etched NSOM probes, the spectra of a thermochromic polymer sample are measured as a function of probe output power, as was previously reported for pulled NSOM probes. The results reveal that sample heating increases rapidly to approximately 55-60 degrees C as output powers reach approximately 50 nW. At higher output powers, the sample heating remains approximately constant up to the maximum power studied of approximately 450 nW. The sample heating profiles measured for etched NSOM probes are consistent with those previously measured for NSOM probes fabricated using the pulling method. At high powers, both pulled and etched NSOM probes fail as the aluminum coating is damaged. For probes fabricated in our laboratory we find failure occurring at input powers of 3.4+/-1.7 and 20.7+/-6.9 mW for pulled and etched probes, respectively. The larger half-cone angle for etched probes ( approximately 15 degrees for etched and approximately 6 degrees for pulled probes) enables more light delivery and also apparently leads to a different failure mechanism. For pulled NSOM probes, high resolution images of NSOM probes as power is increased reveal the development of stress fractures in the coating at a taper diameter of approximately 6 microm. These stress fractures, arising from the differential heating expansion of the dielectric and the metal coating, eventually lead to coating removal and probe failure. For etched tips, the absence of clear stress fractures and the pooled morphology of the damaged aluminum coating following failure suggest that thermal damage may cause coating failure, although other mechanisms cannot be ruled out.

Recent Advances in Microscale Western Blotting.

Western blotting is a ubiquitous tool used extensively in the clinical and research settings to identify proteins and characterize their levels. It has rapidly become a mainstay in research laboratories due to its specificity, low cost, and ease of use. The specificity arises from the orthogonal processes used to identify proteins. Samples are first separated based on size and then probed with antibodies specific for the protein of interest. This confirmatory approach helps avoid pitfalls associated with antibody cross-reactivity and specificity issues. While the technique has evolved since its inception, the last decade has witnessed a paradigm shift in Western blotting technology. The introduction of capillary and microfluidic platforms has significantly decreased time and sample requirements while enabling high-throughput capabilities. These advances have enabled Western analysis down to the single cell level in highly parallel formats, opening vast new opportunities for studying cellular heterogeneity. Recent innovations in microscale Western blotting are surveyed, and the potential for enhancing detection using advances in label-free biosensing is briefly discussed.

Advanced Age Alters Monocyte and Macrophage Responses.

SIGNIFICANCE: With the growing population of baby boomers, there is a great need to determine the effects of advanced age on the function of the immune system. Recent Advances: It is universally accepted that advanced age is associated with a chronic low-grade inflammatory state that is referred to as inflamm-aging, which alters the function of both immune and nonimmune cells. Mononuclear phagocytes play a central role in both the initiation and resolution of inflammation in multiple organ systems and exhibit marked changes in phenotype and function in response to environmental cues, including the low levels of pro-inflammatory mediators seen in the aged. CRITICAL ISSUES: Although we know a great deal about the function of immune cells in young adults and there is a growing body of literature focusing on aging of the adaptive immune system, much less is known about the impact of age on innate immunity and the critical role of the mononuclear phagocytes in this process. FUTURE DIRECTIONS: In this article, there is a focus on the tissue-specific monocyte and macrophage subsets and how they are altered in the aged milieu, with the hope that this compilation of observations will spark an expansion of research in the field. Antioxid. Redox Signal. 25, 805-815.