Analysis of water-soluble vitamins in biopharma raw materials by electrophoresis micro-chips with contactless conductivity detection

Detailed information concerning the composition of the raw materials employed in the production of biologics is important for the efficient control and optimization of bioprocesses. The analytical methods used in these applications must be simple and fast as well as be easily transferable from one site to another. In that context, microchip‐based electrophoresis represents a promising tool for application in the analysis of raw materials in biologics. Using electrophoresis micro‐chips, analysis times can be reduced to seconds and high separation efficiencies can be achieved using extremely low volume samples, minimal reagent consumption and waste generation, low cost/disposability, portability and ease of mass‐production [1]. Additionally the use of Capacitively Coupled Contactless Conductivity Detection (C4D) offers a rather simple and yet sensitive method for detection of ionic species. Recently, C4D has gained much popularity as on‐chip detection in electrophoresis micro‐chips [2]. The main reason for this is that there is no physical contact of the detection electrodes with the electrolyte solution. Therefore, the integration of this detection mode within the analytical system is rather simple. Furthermore, the background noise is significantly reduced leading to lower detection limits than the conventional contact conductivity detection. Vitamins are present at very low concentrations in biopharma raw materials and are usually determined using HPLC and CE methods [3]. Electrophoresis micro‐chips are a very good alternative to these techniques due to the shorter analysis time and yet very good resolution, among others. In this paper, we present the application of electrophoresis micro‐chips with C4D detection to the analysis of water‐soluble vitamins in raw materials used for the production of biologics in bioreactors. For that purpose, hybrid PDMS/glass chips were fabricated by using standard photolithographic techniques (Figure 1). The chip structure contains an extremely long channel of 101 mm (50 x 50 μm width x depth). Figure 2 shows the setup used for vitamins detection

Analysis of biopharma raw materials by electrophoresis microchips with contactless conductivity detection

Detailed information concerning the composition of the raw materials employed in the production of biologics is important for the efficient control and optimization of bioprocesses. We demonstrate the application of electrophoresis microchips with capacitively-coupled contactless conductivity detection (C4D) to the analysis of wa-ter-soluble vitamins and metal cations in raw material solutions that are subse-quently fed into bioreactors for the production of biologics

Liquid recirculation in microfluidic channels by the interplay of capillary and centrifugal forces

We demonstrate a technique to recirculate liquids in a microfluidic device, maintaining a thin fluid layer such that typical diffusion times for analytes to reach the device surface are < 1 min. Fluids can be recirculated at least 1000 times across the same surface region, with no change other than slight evaporation, by alternating the predominance of centrifugal and capillary forces. Mounted on a rotational platform, the device consists of two hydrophilic layers separated by a thin pressure-sensitive adhesive (PSA) layer that defines the microfluidic structure. We demonstrate rapid, effective fluid mixing with this device

Monolithic centrifugal microfluidic platform for bacteria capture and concentration, lysis, nucleic-acid amplification, and real-time detection

We report the design, fabrication, and characterization of a polymer centrifugal microfluidic system for the specific detection of bacterial pathogens. This single-cartridge platform integrates bacteria capture and concentration, supernatant solution removal, lysis, and nucleic-acid sequence-based amplification (NASBA) in a single unit. The unit is fabricated using multilayer lamination and consists of five different polymer layers. Bacteria capture and concentration are accomplished by sedimentation in five minutes. Centrifugation forces also drive the subsequent steps. A wax valve is integrated in the cartridge to enable high-speed centrifugation. Oil is used to prevent evaporation during reactions requiring thermal cycling. Device functionality was demonstrated by real-time detection of E. coli from a 200-muL sample

Development of Innovative Microfluidic Polymeric Technologies for Point-of-care & Integrated Diagnostics Devices

This thesis presents the development of four different microfluidic technologies that can be used as stand-alone devices or integrated in point-of-care systems. The first technology is a rapid, low-cost, portable microfluidic system for assessing the somatic cell count and fat content of milk in 15 min using a “sample-in, answer-out” approach. The system consists of twelve independent microfluidic devices, essentially flattened funnel structures, fabricated on the footprint of a plastic compact disc (CD). The assay separates cells and fat globules based on their densities (by differential sedimentation), concentrating white cells in the closed-end channel near the outer rim of the CD for estimation of total “cell pellet” volume, while fat globules move toward the center of disc rotation, forming a fat “band” in the funnel. The closed-end channel provides accurate cell counts over the range 50,000 to over 3,000,000 cells per mL. A technique is also presented to recirculate liquids in a microfluidic channel by alternating the predominance of centrifugal and capillary forces. With this technique, liquid volumes of μL to mL can be sampled with many sizes of microfluidic channels that contain only a fraction of the sample at one time, provided the channel wall with greatest surface area is hydrophilic. We present a theoretical model describing the balance of centrifugal and capillary forces in the device and validate the model experimentally. Towards the development of an integrated pathogen identification system, two other technologies are demonstrated and implemented. The design, fabrication, and characterization of a polymer centrifugal microfluidic system for the specific detection of bacterial pathogens is presented. This single-cartridge platform integrates bacteria capture and concentration, supernatant solution removal, lysis, and nucleic-acid sequence-based amplification (NASBA) in a single unit. The unit is fabricated using multilayer lamination and consists of five different polymer layers. Bacteria capture and concentration are accomplished by sedimentation in five minutes. Centrifugation forces also drive the subsequent steps. A wax valve is integrated in the cartridge to enable high-speed centrifugation. Oil is used to prevent evaporation during reactions requiring thermal cycling. Device functionality was demonstrated by real-time detection of E. coli cells from a 200-μL sample. Finally, the laser-printer-based fabrication of pressure-resistant microfluidic single-use valves is reported, along with their implementation on pressure-driven and centrifugal microfluidic platforms. A laser printer is used to selectively deposit toner on a plastic substrate in the form of circular dots. After assembly into a microfluidic device, the valve is opened (melted) with a pulse of laser light. This is an easy approach to connect multiple fluidic levels. This simple technology is compatible with a range of polymer microfabrication technologies and should facilitate the development of fully integrated, (re)configurable, and automated lab-on-a-chip systems, particularly when reagents must be stored on chip for extended periods, e.g. for medical diagnostic devices, lab-on-a-chip synthetic systems, or hazardous bio/chemical analysis platforms

A 1024-sample serum analyzer chip for cancer diagnostics

We present a platform that combines microarrays and microfluidic techniques to measure four protein biomarkers in 1024 serum samples for a total of 4096 assays per device. Detection is based on a surface fluorescence sandwich immunoassay with a limit of detection of similar to 1 pM for most of the proteins measured: PSA, TNF-alpha, IL-1 beta, and IL-6. To validate the utility of our platform, we measured these four biomarkers in 20 clinical human serum samples, 10 from prostate cancer patients and 10 female and male controls. We compared the results of our platform to a conventional ELISA and found a good correlation between them. However, compared to a classical ELISA, our device reduces the total cost of reagents by 4 orders of magnitude while increasing throughput by 2 orders of magnitude. Overall, we demonstrate an integrated approach to perform low-cost and rapid quantification of protein biomarkers from over one thousand serum samples. This new high-throughput technology will have a significant impact on disease diagnosis and management

Birefringent Optofluidic Gratings

A set of parallel microfluidic channels behaving as a diffraction grating operating in the Raman-Nath regime has been fabricated and studied. The diffraction efficiency of such structure can be tuned by selecting a liquid with a particular refractive index and/or optical anisotropy. Alternatively the optical properties of the liquid can be characterised by measuring the diffraction efficiency and the state of polarization of the diffracted beam. In this work, the microfluidic channels under study have been filled with penicillin molecules dissolved in water. Due to the chirality of the penicillin, the liquid has been found to have circular birefringence of 2.14 × 10-7. The addition of the anisotropic liquid modifies the polarization properties of the microfluidic diffraction grating. The diffraction efficiency of the grating has been characterised for different probe beam wavelengths and states of polarization. Currently the diffraction efficiency of the device is low - 1.7%, but different approaches for its improvement have been discussed

Pretransplant CMV-Specific T-Cell Immunity But Not Dose of Antithymocyte Globulin Is Associated With Recovery of Specific Immunity After Kidney Transplantation

Background This is a prospective, multicenter, observational study in cytomegalovirus (CMV)-seropositive kidney transplant recipients with pretransplant CMV-specific cell-mediated immunity (CMV-CMI) receiving antithymocyte globulin (ATG). We aimed to investigate posttransplant CMV-CMI over time and the impact of the dose-dependent ATG. Methods CMV-CMI was assessed at days +30, +45, +60, and +90 after transplantation with the QuantiFERON-CMV assay. A reactive result (interferon-γ [IFN-γ] ≥ 0.2 IU/mL) indicated a positive CMV-CMI. Results A total of 78 positive CMV-CMI patients were enrolled in the study, of which 59.5% had a positive CMV-CMI at day +30 and 82.7% at day +90. Multivariate logistic regression analysis showed that ATG dose was not associated with positive CMV-CMI at any point. However, pretransplant IFN-γ level (>12 IU/mL vs ≤12 IU/mL) was associated with positive CMV-CMI at day +30 (odds ratio, 12.9; 95% confidence interval, 3.1–53.3; P < .001). In addition, all the patients who did not recover CMV-CMI at day +90 had a pretransplant IFN-γ level ≤12 IU/mL. Conclusions More than half of CMV-seropositive kidney transplant recipients receiving ATG recover (or maintain) CMV-CMI by the first month after transplantation. The pretransplant IFN-γ level, but not the ATG dose, shows a strong association with the kinetics of this recovery

Visualizing Cancer

Imaging has had a profound impact on our ability to understand and treat cancer. We invited some experts to discuss imaging approaches that can be used in various aspects of cancer research, from investigating the complexity and diversity of cancer cells and their environments to guiding clinical decision-making