Apodization‐Assisted Subdiffraction Near‐Field Localization in 2D Phase Diffraction Grating

Conventional phase diffraction gratings can be used to localize the incoming optical radiation in the near‐field region. A new design of the binary phase diffraction grating is proposed with embedded pupil opaque mask inside each stripe. By means of numerical simulations, it is shown that with this masked phase grating the spatial resolution of the near‐field localization can be substantially improved and brought even beyond the solid immersion limit (λ/2n). Moreover, due to anomalous apodization effect, the subdiffraction field localization is accompanied by intensity enhancement as compared to the non‐masked design. The pupil mask rearranges the optical fluxes within the stripes and promotes the Fano resonances excitation in the periodic step lattice. This can be important for advancing the phase grating‐based super‐resolution technologies, including subdiffraction imaging, interferometry, and surface fabrication.     

Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity

Post-translational modifications (PTMs) of adeno-associated virus (AAV) capsid proteins tune and regulate the AAV infective life cycle, which can impact the safety and efficacy of AAV gene therapy products. Many of these PTMs induce changes in protein charge heterogeneity, including deamidation, oxidation, glycation, and glycosylation. To characterize the charge heterogeneity of a protein, imaged capillary isoelectric focusing (icIEF) has become the gold standard method. We have previously reported an icIEF method with native fluorescence detection for denatured AAV capsid protein charge heterogeneity analysis. Although well suited for final products, the method does not have sufficient sensitivity for upstream, low-concentration AAV samples, and lacks the specificity for capsid protein detection in complex samples like cell culture supernatants and cell lysates. In contrast, the combination of icIEF, protein capture, and immunodetection affords significantly higher sensitivity and specificity, addressing the challenges of the icIEF method. By leveraging different primary antibodies, the icIEF immunoassay provides additional selectivity and affords a detailed characterization of individual AAV capsid proteins. In this study, we describe an icIEF immunoassay method for AAV analysis that is 90 times more sensitive than native fluorescence icIEF. This icIEF immunoassay provides AAV stability monitoring, where changes in individual capsid protein charge heterogeneity can be observed in response to heat stress. When applied to different AAV serotypes, this method also provides serotype identity with reproducible quantification of VP protein peak areas and apparent isoelectric point (pI). Overall, the described icIEF immunoassay is a sensitive, reproducible, quantitative, specific, and selective tool that can be used across the AAV biomanufacturing process, especially in upstream process development where complex sample types are often encountered.     

Ion transmission in an electrospray ionization-mass spectrometry interface using an S-lens

We present the design and performance of an in-house built electrospray ionization-mass spectrometry (ESI-MS) interface equipped with an S-lens ion guide. The ion source was designed specifically for our ion beam experiments to investigate the chemical reactivity and deposition of the clusters and nanoparticles. It includes standard ESI-MS interface components, such as nanoelectrospray, ion transfer capillary, and the S-lens. A custom design enables systematic optimization of all relevant factors influencing ion formation and transfer through the interface. By varying the ESI voltage and flow rate, we determined the optimal operating conditions for selected silica emitters. A comparison of the pulled silica emitters with different tip inner diameters reveals that the total ion current is highest for the largest tip, whereas a tip with the smallest diameter exhibited the highest transmission efficiency through the ESI-MS interface. Ion transmission through the transfer capillary is strongly limited by its length, but the loss of ions can be reduced by increasing the capillary voltage and temperature. The S-lens was characterized over a wide range of RF frequencies and amplitudes. Maximum ion current was detected at RF amplitudes greater than 50 V peak-to-peak (p/p) and frequencies above 750 kHz, with a stable ion transmission region of about 20%. A factor of 2.6 increase in total ion current is observed for 650 kHz as RF amplitudes reach 400 V p/p. Higher RF amplitudes also focus the ions into a narrow beam, which mitigates their losses when passing through the ion guide.     

Application of a multivariate approach for analyte focusing by micelle collapse‐micellar electrokinetic chromatography for analyzing sunscreen agents in cosmetics

The operating parameters that affect the performance of the online preconcentration technique “analyte focusing by micelle collapse‐MEKC (AFMC‐MEKC)” were examined using a multivariate approach involving experimental design to determine the sunscreen agents in cosmetics. Compared to the single‐variable approach, the advantage of the multivariate approach was that many factors could be investigated simultaneously to obtain the best separation condition. A fractional factorial design was used to identify the fewest significant factors in the central composite design (cCD). The cCD was adopted for evaluating the location of the minimum or maximum response in this study. The influences of the experimental variables on the response were investigated by applying a chromatographic exponential function. The optimized condition and the relationship between the experimental variables were acquired using the JMP software. The ANOVA analysis indicated that the Tris pH value, SDS concentration, and ethanol percentage influenced the separation quality and significantly contributed to the model. The optimized condition of the running buffer was 10 mM Tris buffer (pH 9.5) containing 60 mM SDS, 7 mM γ‐CD, and 20% v/v ethanol. The sample was prepared in 100 mM Tris buffer (pH 9.0) containing 7.5 mM SDS and 20% v/v ethanol. The SDS concentration in the sample matrix was slightly greater than the CMC value that makes the micelle be easily collapsed and the analytes be accumulated in the capillary. In addition, sunscreen agents in cosmetics after 1000‐fold dilution were successfully determined by AFMC‐MEKC.     

Analysis of genetic variants of transferrin in human serum after desialylation by capillary zone electrophoresis and capillary isoelectric focusing

Capillary electrophoresis analysis of transferrin in human serum is used to assess genetic variants after desialylation with neuraminidase and iron saturation to reduce the complexity of the transferrin pattern and thus facilitate the recognition of transferrin polymorphisms. Asialo‐transferrin forms are analyzed by capillary zone electrophoresis using assay conditions as for the monitoring of carbohydrate‐deficient transferrin or by capillary isoelectric focusing in a pH 5–8 gradient which requires immunoextraction of transferrin prior to analysis. With the carrier ampholytes used, peaks for iron saturated and iron depleted transferrin are monitored which indicates complexation of iron ions by carrier ampholytes. For BC, CD, and BD genetic variants, the expected peaks for B, C, and D forms of transferrin were detected with both methods. Monitoring of CC patterns revealed three cases, namely those producing double peaks in both methods, a double peak in capillary isoelectric focusing only and a double peak in capillary zone electrophoresis only. For all samples analyzed, data obtained by capillary isoelectric focusing could be confirmed with gel isoelectric focusing. The two capillary electrophoresis methods are shown to represent effective tools to assess unusual transferrin patterns, including genetic variants with dissimilar abundances of the two forms.     

Advanced online mass spectrometry detection of proteins separated by capillary isoelectric focusing after sequential injection

Capillary isoelectric focusing hyphenated with mass spectrometry detection, following the sequential injection of the carrier ampholytes and the sample zone, is highly efficient for the characterization of proteins. The main advantage of the sequential injection protocol is that ampholytes, with pH ranges, which are not supposed to cover the isoelectric points of the sample components, can be used for separation. The method then allows online mass spectrometry detection of separated analytes either in the absence (substances that have left the pH gradient) or in the presence of low‐level ampholytes (substances that are migrating within the pH gradient). The appearance of the substances within, or outside the pH gradient depends on, e.g., the composition of the ampholytes (broad or narrow pH range) or on the composition of electrolyte solutions. The experiments performed in coated capillaries (with polyvinyl alcohol or with polyacrylamide) show that the amount and the injection length of the ampholytes influence the length of the pH gradient formed in the capillary.     

Quantification of the bisphosphonate alendronate using capillary electrophoresis mass spectrometry with dynamic pH barrage junction focusing

A quantitative method was developed for the direct identity confirmation and quantification of alendronate using CE-MS combined with a pH-assisted focusing technique, dynamic pH barrage junction focusing. A pH-induced variation in electrophoretic mobility led to online focusing of alendronate at the sample/pH barrage boundary, significantly improving the detection sensitivity. In addition, the use of a flow-through microvial CE electrospray interface and the multiple reaction monitoring mode of MS further improved the specificity and quantification capability of this technology. This quantitative method presented a wide linear dynamic range over 8–2000 ng/mL and an LOD of 2 ng/mL. A 460-fold improvement in sensitivity was obtained when pH barrage junction focusing was applied during the CE process, in comparison to when normal CE was conducted without online sample stacking. The superior detection sensitivity over previously reported methods enables direct analysis of bisphosphonate compounds, eliminating tedious pre-column sample enrichment and derivatization. Validation of alendronate content in a commercial drug tablet further proved the reliability and power of this method. This simple method with no sample derivatization, superior sensitivity, and short run time (<8 min) is a promising alternative for accurate quantification of alendronate and other types of bisphosphonate compounds in both drug formulations and plasma samples.     

Secondary-flow-aided single-train elastic-inertial focusing in low elasticity viscoelastic fluids

Elastic-inertial focusing has attracted increasing interest in recent years due to the three-dimensional (3D) single-train focusing ability it offers. However, multi-train focusing, instead of single-train focusing, was observed in viscoelastic fluids with low elasticity as a result of the competition between inertia effect and viscoelasticity effect. To address this issue, we employed the secondary flow to facilitate single-train elastic-inertial focusing in low elasticity viscoelastic fluids. A three-section contraction-expansion channel was designed to induce the secondary flow to pinch the multiplex focusing trains into a single one exactly at the channel centerline. After demonstrating the focusing process and mechanism in our device, we systematically explored and discussed the effects of particle diameter, operational flow rate, polymer concentration, and channel dimension on particle focusing performances. Our device enables single-train focusing of particles in viscoelastic fluids with low elasticity, and offers advantages of planar single-layer structure, and sheathless, external-field free operation.