Hadewych Halewyck

Microfluidics in macro-biomolecules analysis: macro inside in a nano world

Use of microfluidic devices in the life sciences and medicine has created the possibility of performing investigations at the molecular level. Moreover, microfluidic devices are also part of the technological framework that has enabled a new type of scientific information to be revealed, i.e. that based on intensive screening of complete sets of gene and protein sequences. A deeper bioanalytical perspective may provide quantitative and qualitative tools, enabling study of various diseases and, eventually, may offer support for the development of accurate and reliable methods for clinical assessment. This would open the way to molecule-based diagnostics, i.e. establish accurate diagnosis and disease prognosis based on identification and/or quantification of biomacromolecules, for example proteins or nucleic acids. Finally, the development of disposable and portable devices for molecule-based diagnosis would provide the perfect translation of the science behind life-science research into practical applications dedicated to patients and health practitioners. This review provides an analytical perspective of the impact of microfluidics on the detection and characterization of bio-macromolecules involved in pathological processes. The main features of molecule-based diagnostics and the specific requirements for the diagnostic devices are discussed. Further, the techniques currently used for testing bio-macromolecules for potential diagnostic purposes are identified, emphasizing the newest developments. Subsequently, the challenges of this type of application and the status of commercially available devices are highlighted, and future trends are noted.

Mechanism of Action and Capsid-Stabilizing Properties of VHHs with an In Vitro Antipolioviral Activity

ABSTRACT Previously, we reported on the in vitro antiviral activity of single-domain antibody fragments (VHHs) directed against poliovirus type 1. Five VHHs were found to neutralize poliovirus type 1 in an in vitro setting and showed 50% effective concentrations (EC50s) in the nanomolar range. In the present study, we further investigated the mechanism of action of these VHHs. All five VHHs interfere at multiple levels of the viral replication cycle, as they interfere both with attachment of the virus to cells and with viral uncoating. The latter effect is consistent with their ability to stabilize the poliovirus capsid, as observed in a ThermoFluor thermal shift assay, in which the virus is gradually heated and the temperature causing 50% of the RNA to be released from the capsid is determined, either in the presence or in the absence of the VHHs. The VHH-capsid interactions were also seen to induce aggregation of the virus-VHH complexes. However, this observation cannot yet be linked to their mechanism of action. Cryo-electron microscopy (cryo-EM) reconstructions of two VHHs in complex with poliovirus type 1 show no conformational changes of the capsid to explain this aggregation. On the other hand, these reconstructions do show that the binding sites of VHHs PVSP6A and PVSP29F overlap the binding site for the poliovirus receptor (CD155/PVR) and span interfaces that are altered during receptor-induced conformational changes associated with cell entry. This may explain the interference at the level of cell attachment of the virus as well as their effect on uncoating. IMPORTANCE The study describes the mechanism of neutralization and the capsid-stabilizing activity of five single-domain antibody fragments (VHHs) that have an in vitro neutralizing activity against poliovirus type 1. The results show that the VHHs interfere at multiple levels of the viral replication cycle (cell attachment and viral uncoating). These mechanisms are possibly shared by some conventional antibodies and may therefore provide some insight into the natural immune responses. Since the binding sites of two VHHs studied by cryo-EM are very similar to that of the receptor, the VHHs can be used as probes to study the authentic virus-cell interaction. The structures and conclusions in this study are original and raise interesting findings regarding virus-receptor interactions and the order of key events early in infection.

Improving the capillary electrophoretic analysis of poliovirus using a Plackett-Burman design.

Separation techniques may offer interesting alternatives to classical virological techniques both for fundamental research purposes and for vaccine manufacturing. A capillary electrophoretic method for the analysis of the poliovirus was developed based on conditions for the human rhinovirus taken from literature. The method was optimized using a 12-experiment Plackett-Burman design, applied in order to examine simultaneously the effects of eight factors on responses such as, mobility of the electroosmotic flow, effective mobility of the poliovirus, analysis time and resolution between the virus peak and a system peak. The proposed method manages to perform an acceptable separation of poliovirus particles using a 50 mM borate buffer with 25 mM SDS, in an uncoated fused-silica capillary upon application of 10 kV at 30 degrees C. The linearity of the proposed method was investigated for a range of poliovirus dilutions up to 140 microg/mL.

Identification of poliovirions and subviral particles by capillary electrophoresis

Poliovirions, purified from infected cell extracts with anion‐exchange chromatography, can be analyzed and identified by CE in untreated fused silica capillaries using UV detection. Other subviral particles can be eluted as well from the same infected cell extract using a higher salt concentration buffer on the ion‐exchange chromatography. Virions can be identified because of their conversion into empty capsids upon heating at 56°C. As a result of heating, the viral genome is released from the capsid. Here, we show that during this incubation some intermediate particles were found. The latter were identified by enzymatic peak shift analysis. The high salt concentration eluate subviral particles were analyzed with preincubation affinity CE together with their sensitivity for RNase and proteinase K treatment. Electropherograms of the higher salt concentration eluate display a mixture of at least four different subviral particles. One particle proved to have an [N1, H] antigenicity and was resistant to RNase and proteinase K digestion. The remaining particles were all sensitive to proteinase K treatment. This CE method proved to be valuable in the detection, identification and analysis of poliovirions and poliovirus particles offering an alternative powerful, cheap, fast and easy analysis method.

Rational use of stacking principles for signal enhancement in capillary electrophoretic separations of poliovirus samples.

The use of an earlier developed capillary electrophoresis (CE) method, either to investigate poliovirus (PV) samples with a low viral-purity level or to study the less abundant sub-viral particles, revealed the necessity for an intra-column signal enhancement strategy. Although intra-column signal enhancement is a very popular approach to assay small molecules, it is less straightforward for the analysis of biological macromolecules or particles. A reason could be that, for a proper signal enhancement approach, these samples have to be thoroughly studied to understand the factors affecting the separation process. For the investigated PV samples, a screening design revealed that injecting larger sample plugs significantly enhanced the analytical signal, but also significantly decreased the separation efficiency. A subsequently executed central composite design determined the largest sample plug that can be injected without compromising the separation. Finally, the sample dilution and the length of the injected plug were used for tuning the intensity of the analytical response. Two combinations of sample dilution and injected plug size, at extreme values, were investigated in detail to define the best procedure for PV analysis using CE. In both situations, PV was effectively separated and quantified in rather complex samples, showing a good repeatability, an acceptable linearity for the PV particles and a decreased limit of detection in comparison with the existing method. In conclusion, intra-column signal enhancement can be successfully applied for viral suspensions, extending the applicability of CE methods to samples with lower virus concentrations, and/or allowing a significant reduction in the minimum required volume of sample. For PV samples, 5μl of sample is necessary instead of the previous 20μl, while the analytical signal was enhanced up to 14 times. The results of this study can provide a basis for the development of routine CE methods for viral particle analysis, especially when rational and reproducible signal enhancement is required.

Poliovirus separation from cell extracts using capillary electrophoresis: Potential use in vaccine production and control?

Rapid assessment of the concentration of virus particles in a given sample remains a challenge. Modern separation methods, such as capillary electrophoresis, were proposed recently to study viruses and viral infection or to separate and characterize viral vaccines in a time-efficient manner. Even though capillary electrophoresis is much more rapid than traditional virological methods and has the advantages of automation, increased precision and reliability, it has the drawback of reduced sensitivity for low concentrations. A sensitivity improvement is then necessary in many cases for a successful application. However, to date, only highly purified viral samples were examined using capillary electrophoresis. The injection of larger sample volumes, followed by intra-capillary concentration, was used in this study for cell extracts. Poliovirus was successfully detected rapidly, without any laborious staining procedures and incubation times. The method is simple, fast, automatic, requires only minute amounts of samples and reagents, and no expensive dyes or biological reagents. Additionally, the method showed a potential for monitoring the viral load during growth and purification, with obvious prospects for the optimization of the variable and time-consuming virus propagation procedures. The results of this study provide a potential basis for the development of routine methods for viral particles analysis, irrespective of their infective properties. In the future, the capillary electrophoresis test could help study the relationship between the intact poliovirus particles and the D-antigenic properties of a viral suspension, or could represent a supplementary or alternative test for virus concentration and D-antigen assays during vaccine production.

Affinity capillary electrophoresis to evaluate the complex formation between poliovirus and nanobodies

It was demonstrated that nanobodies with an in vitro neutralizing activity against poliovirus type 1 interact with native virions. Here, the use of capillary electrophoresis was investigated as an alternative technique for the evaluation of the formation of nanobody-poliovirus complexes, and therefore predicting the in vitro neutralizing activity of the nanobodies. The macromolecules are preincubated offline in a specific nanobody-to-virus ratio and analyzed by capillary electrophoresis with UV detection. At low nanobody-to-virus ratios, a clear shift in migration time of the viral peak was observed. A broad peak was obtained, indicating the presence of a heterogeneous population of nanobody-virion complexes, caused by the binding of different numbers of nanobodies to the virus particle. At elevated nanobody-to-virus ratios, a cluster of peaks appeared, showing an additional increase in migration times. It was shown that, at these high molar excesses, aggregates were formed. The developed capillary electrophoresis method can be used as a rapid, qualitative screening for the affinity between poliovirus and nanobodies, based on a clearly visible and measurable shift in migration time. The advantages of this technique include that there is no need for antigen immobilization as in enzyme-linked immunosorbent assays or surface plasmon resonance for the use of radiolabeled virus or for the performance of labor- and time-intensive plaque-forming neutralization assays.

The effect of anionic surfactant on poliovirus particles during capillary electrophoresis.

Because of its essential role in SDS-PAGE, sodium dodecylsulphate (SDS) is generally associated with protein denaturation. However, for SDS-PAGE, proteins are linearized in the presence of SDS, following the exposure to high temperatures and reducing agents. In comparison, the conditions employed during a capillary electrophoretic (CE) separation involve only a limited exposure to SDS, at much lower temperatures. As the outer surface of the non-enveloped viruses consists of proteins, virus interaction with SDS can be judged from the perspective of SDS-protein interaction. Several studies have indicated that proteins have a different susceptibility to SDS, depending on their secondary structure and number of subunits. Therefore it is not straightforward to estimate what should be expected when intact polioviruses and subviral particles obtained by thermal conversion of the poliovirions, are exposed to SDS during CE separation. In this study it is shown that, during CE separations, SDS has no effect on the integrity of the poliovirion, but the presence of SDS in the separation system influences the poliovirus peak height and shape. The implication of SDS in the CE separation of poliovirus is discussed in detail. On the contrary, the proteinaceous subviral particles, such as the empty capsids, are less stable in the presence of SDS during the CE separation, and aggregates between the individual poliovirus capsid proteins and SDS are formed. Finally, we have proposed an alternative separation approach, involving an SDS gradient, for an improved separation of the subviral particles.

Impact of the sample matrix composition on the signal enhancement in the capillary electrophoretic separation of poliovirus samples.

The development of capillary electrophoretic applications aiming to provide reliable stability assessment of viral suspensions, to detect subviral particles from cell extracts or to study the interactions between virus particles and various biomolecules, cannot be done without a thorough understanding of the sample matrix contribution to the observed electrophoretic behaviour. The present study thoroughly investigates the effect of the sample matrix on the electrophoretic behaviour of poliovirus injected as sample plugs of 1%, 5% and 12% effective capillary length. The effect of the sample matrix for three different poliovirus batches was evaluated. Additionally, simulated samples, obtained from concentrated poliovirus suspensions of high purity and diluted with commonly used lab buffers in order to obtain samples with either high or low conductivities, were also investigated. The goal of the study was to obtain a better understanding of the effect of the sample matrix on the signal enhancement, in order to define a general approach allowing a repeatable capillary electrophoretic (CE) separation of poliovirus from complex samples. This study clearly demonstrates that the sample matrix has an important influence on the sensitivity of the CE poliovirus separations. Translation of these observations into routine practice involves several compromises and a set of rules in order to reduce day-to-day variation and to maximize sensitivity.