Magnetic particle plug-based assays for biomarker analysis

Conventional immunoassays offer selective and quantitative detection of a number of biomarkers, but are laborious and time-consuming. Magnetic particle-based assays allow easy and rapid selection of analytes, but still suffer from the requirement of tedious multiple reaction and washing steps. Here, we demonstrate the trapping of functionalised magnetic particles within a microchannel for performing rapid immunoassays by flushing consecutive reagent and washing solutions over the trapped particle plug. Three main studies were performed to investigate the potential of the platform for quantitative analysis of biomarkers: (i) a streptavidin-biotin binding assay; (ii) a sandwich assay of the inflammation biomarker, C-reactive protein (CRP); and (iii) detection of the steroid hormone, progesterone (P4), towards a competitive assay. Quantitative analysis with low limits of detection was demonstrated with streptavidin-biotin, while the CRP and P4 assays exhibited the ability to detect clinically relevant analytes, and all assays were completed in only 15 min. These preliminary results show the great potential of the platform for performing rapid, low volume magnetic particle plug-based assays of a range of clinical biomarkers via an exceedingly simple technique

Micro- and Nanofluidics for Bionanoparticle Analysis

Bionanoparticles such as microorganisms and exosomes are recoganized as important targets for clinical applications, food safety, and environmental monitoring. Other nanoscale biological particles, includeing liposomes, micelles, and functionalized polymeric particles are widely used in nanomedicines. The recent deveopment of microfluidic and nanofluidic technologies has enabled the separation and anslysis of these species in a lab-on-a-chip platform, while there are still many challenges to address before these analytical tools can be adopted in practice. For example, the complex matrices within which these species reside in create a high background for their detection. Their small dimension and often low concentration demand creative strategies to amplify the sensing signal and enhance the detection speed. This Special Issue aims to recruit recent discoveries and developments of micro- and nanofluidic strategies for the processing and analysis of biological nanoparticles. The collection of papers will hopefully bring out more innovative ideas and fundamental insights to overcome the hurdles faced in the separation and detection of bionanoparticles

Study of methods for platelet function testing in the perspective of lab-on-chip applications

Research goals Platelets are reactive cells with the main function to maintain blood vessel integrity. Altered platelet function can lead to cardiovascular diseases such as thrombosis or bleeding disorders and platelets can also play a role in atherosclerosis. Current methods to quantify platelet function are laboratory techniques such as flow cytometry, aggregometry or luminescent assays. However, these techniques are complex and time consuming. Therefore we are interested in novel methods suited for future application in a lab-on-a-chip format. In our research we have focused on the use of well-established biomarkers, such as membrane marker expression, cytosolic calcium signaling and secretion markers, to quantify platelet activation as well as responsiveness. We report studies on platelet function using these read-out parameters and we discuss the relevance of the results for lab-on-a-chip biosensor research. Measurement of platelet membrane markers using antibody coated magnetic beads Magnetic beads are convenient carriers for rapid capture and manipulation of biological cells in a miniaturized system. We have studied the use of antibody coated magnetic beads to measure platelet function via membrane markers expressed upon activation. We used anti-P-selectin coated beads to capture activated platelets from samples stimulated with Thrombin Receptor Activator Peptide (TRAP). The responsiveness of the platelets was analyzed via the remaining unbound platelets in solution and compared to a reference method in which the number of activated platelets is analyzed via fluorescent labeling. The effective concentration for platelet responsiveness found with the bead capture assay (17.9 ± 4.9 µM) was in good agreement with the effective concentration found with the reference assay (23.5 ± 0.4 µM) in buffer. In 10% plasma the effective concentrations were 14.0 ± 4.4 µM and 13.8 ± 0.3 µM respectively, proving that platelet responsiveness can be quantified using antibody coated magnetic beads. In addition, we showed that we were able to discriminate between non-specific and specific interactions between functionalized beads and immobilized platelet with the use of magnetic actuation. The demonstrated read-out method is an interesting first step toward a lab-on-a-chip system for platelet function testing. Measurement of calcium signaling to study platelet-surface interactions Most lab-on-chip biosensor concepts are based on the use of a substrate for immobilization and subsequent detection of the biological system of interest. So for lab-on-chip platelet function testing it is important to understand the influence that a substrate can have on the platelets. We studied the interaction between platelets and various surfaces with calcium signaling in platelets. We used a calcium indicator and studied the response of individual platelets upon adhesion to Bovine Serum Albumin (BSA), Poly-L-lysine (PLL), mouse immunoglobulin (IgG), anti-GPIb and collagen coated surfaces. We recorded the fraction of cells that increase their cytosolic calcium upon binding to these surfaces. Furthermore, we recorded the delay time between the moments of platelet adhesion or chemical stimulation and the increase of cytosolic calcium. The experiments showed binding of platelets to PLL, IgG, anti-GP1b and collagen, but not to BSA coated surfaces. We found that under static conditions the number of cells that respond upon binding to an IgG coated surface (40 ± 7 % with Fc-receptor blocker; 41 ± 6 % without Fcreceptor blocker) was the lowest, followed by adhesion on a PLL coated surface (74 ± 7 %). On an anti-GPIb (88 ± 3 %) or a collagen coated surface (89 ± 4 %), the percentage of responding cells was similar. In addition, we found that the percentage of responding immobilized cells on a chemical stimulus was the lowest on anti-GPIb (7 ± 4 %) or collagen (6 ± 4 %), followed by an IgG coated surface with Fc-receptor blocker (39 ± 3 %) or without Fc-receptor blocker (35 ± 2 %). The percentage of cells responding to chemical stimulation was the highest on a BSA coated surface (95 ± 4 %). These measurements showed that there is a negative correlation between the percentage of cells that respond upon binding and that respond to chemical stimulation after binding. The delay times found upon binding ranged from 11 ± 8 s for platelets on PLL, followed by an anti-GPIb (29 ± 19 s) or collagen (24 ± 21 s) coated surface, and IgG coated surfaces with Fc-receptor blocker (62 ± 19 s) and without Fc-receptor blocker (51 ± 26 s). The delay times recorded on chemical stimulation were probably not sensitive enough to measure the physiological processes in adhered platelets. From the data we conclude that BSA is the most quiescent surface for platelet immobilization, while an anti-GPIb coated surface showed to be as thrombogenic as a collagen coated surface. Our experiments demonstrate that the analysis of the responding cells upon binding and chemical stimulation can discriminate between different types of platelet-surface interactions. We expect that the discrimination can be further improved e.g. by automated data processing. In conclusion, we find that the measurement of the responding fraction and the response delay time by calcium signaling are convenient methods to study the time-dependent interaction of platelets with surfaces relevant for lab-on-chip applications. Measurement of exocytosis to study platelet-surface interactions In our second approach to investigate the interaction between platelets and surfaces, we have studied the secretion process of dense granules of a cell ensemble, as well as single cell exocytosis. Adenosine triphosphate (ATP) secretion was quantified by the luminescent luciferin/luciferase reaction. Platelets were allowed to interact with BSA, PLL, mouse IgG and anti-GPIb coated surfaces, and after incubation the amount of secreted ATP was analyzed. The baseline signal was given by ATP secretion from resting platelets in suspension. ATP levels secreted from platelets immobilized on BSA were approximately 2 times as high as the baseline. The immobilization of platelets on PLL showed about 4 times baseline ATP concentrations. On IgG as well as anti-GPIb the maximum amount of ATP was secreted after 1 hour of platelet incubation. Again, we can conclude that the most quiescent surface for platelet immobilization is BSA, followed by PLL, mouse IgG and anti-GPIb. We have also studied the immobilization of the enzyme luciferase on a surface, in order to enable the detection of ATP direct at a surface. We have demonstrated that luciferase adsorbed onto PLL is indeed active and generates luminescence in the presence of ATP. However, the assay still has a low sensitivity and needs further optimization. Finally, we have investigated an exploratory method to resolve exocytosis in single platelet cells, using the fluorescent staining of the dense granules by the quinacrine dye. In our experiments we have observed that the exocytosis events are induced by the fluorescence excitation light. Very probably this is caused by the production of reactive oxygen, which disintegrates the vesicle membrane thereby releasing the vesicle content. Further studies may focus on the use of lower quinacrine concentrations, the use of lower light intensities, or on ways to scavenge reactive oxygen. It will be interesting to further develop the exocytosis assay, as a quantitative method for research on biosensor assays and surfaces suitable for biosensor applications. Conclusions and outlook The scope of the research presented in this thesis was to develop knowledge to support future technological developments, aiming at the measurement of platelet activation or responsiveness in a lab-on-chip device. We found that magnetic particles functionalized with specific antibodies can be used to measure platelet responsiveness. We have also studied the interaction of platelet with different surfaces, by an intracellular calcium signaling assay and by an ATP exocytosis assay using luciferin/luciferase. The most quiescent surface for platelets was BSA, but this surface has a low binding affinity for platelets. Anti-GPIb and collagen coated surfaces show stronger binding, but these surfaces change the activation status of the platelets. In view of these results, we have proposed a new concept to measure platelet function in a lab-on- chip device, namely by labelling platelet activation markers prior to platelet immobilization on a surface. We envision that this design will enable the sensitive labelling of platelets as well as the accurate readout of the platelets at a detection surface in the lab-on-chip device

Advances in Microfluidics and Lab-on-a-Chip Technologies

Advances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains such as biology research, infectious disease management, food safety, and biodefense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care. It has also introduced new functionality, enhanced sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods such as nucleic acid amplification, next-generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities

A novel blood proteomic signature for prostate cancer

International audienceSimple Summary Despite intensive research, effective tools for detection and monitoring of prostate cancer remain to be found. Prostate-specific antigen (PSA), commonly used in prostate cancer assessments, can lead to overdiagnosis and overtreatment of indolent disease. This highlights the need for supporting non-invasive diagnostic, prognostic, and disease stratification biomarkers that could complement PSA in clinical decision-taking via increased sensitivity and specificity. In order to address this need, we uncover novel prostate cancer protein signatures by leveraging a cutting-edge analytical technique to measure proteins in patient samples. This strategy was used as a discovery tool to identify changes in protein levels in the serum of newly diagnosed patients as compared with healthy controls; the feature set was then further validated by reference to a second cohort of patients, achieving a high discriminatory ability. The proteomic maps generated also identified relevant changes in biological functions, notably the complement cascade. Prostate cancer is the most common malignant tumour in men. Improved testing for diagnosis, risk prediction, and response to treatment would improve care. Here, we identified a proteomic signature of prostate cancer in peripheral blood using data-independent acquisition mass spectrometry combined with machine learning. A highly predictive signature was derived, which was associated with relevant pathways, including the coagulation, complement, and clotting cascades, as well as plasma lipoprotein particle remodeling. We further validated the identified biomarkers against a second cohort, identifying a panel of five key markers (GP5, SERPINA5, ECM1, IGHG1, and THBS1) which retained most of the diagnostic power of the overall dataset, achieving an AUC of 0.91. Taken together, this study provides a proteomic signature complementary to PSA for the diagnosis of patients with localised prostate cancer, with the further potential for assessing risk of future development of prostate cancer. Data are available via ProteomeXchange with identifier PXD025484

DEVELOPMENT OF MICROFLUIDIC PLATFORMS AS A TOOL FOR HIGH-THROUGHPUT BIOMARKER SCREENING

Droplet microfluidic platforms are in the early stages of revolutionizing high throughput and combinatorial sample screening for bioanalytical applications. However, many droplet platforms are incapable of addressing the needs of numerous applications, which require high degrees of multiplexing, as well as high-throughput analysis of multiple samples. Examples of applications include single nucleotide polymorphism (SNP) analysis for crop improvement and genotyping for the identification of genes associated with common diseases. My PhD thesis focused on developing microfluidic devices to extend their capabilities to meet the needs of a wide array of applications

Porous Bead-Based Diagnostic Platforms: Bridging the Gaps in Healthcare

Advances in lab-on-a-chip systems have strong potential for multiplexed detection of a wide range of analytes with reduced sample and reagent volume; lower costs and shorter analysis times. The completion of high-fidelity multiplexed and multiclass assays remains a challenge for the medical microdevice field; as it struggles to achieve and expand upon at the point-of-care the quality of results that are achieved now routinely in remote laboratory settings. This review article serves to explore for the first time the key intersection of multiplexed bead-based detection systems with integrated microfluidic structures alongside porous capture elements together with biomarker validation studies. These strategically important elements are evaluated here in the context of platform generation as suitable for near-patient testing. Essential issues related to the scalability of these modular sensor ensembles are explored as are attempts to move such multiplexed and multiclass platforms into large-scale clinical trials. Recent efforts in these bead sensors have shown advantages over planar microarrays in terms of their capacity to generate multiplexed test results with shorter analysis times. Through high surface-to-volume ratios and encoding capabilities; porous bead-based ensembles; when combined with microfluidic elements; allow for high-throughput testing for enzymatic assays; general chemistries; protein; antibody and oligonucleotide applications

Microfluidic Immunoassays Based on Self-Assembled Magnetic Bead Patterns and Time-Resolved Luminescence Detection

Microfluidic bio-assays have emerged as the most privileged solutions and provide the basis for the realization of miniaturized bio-analytical systems and clinical diagnostic devices that are portable, user-friendly and cost-effective (Lab-on-a-chip). Two important steps that are implemented in a microfluidic bio-assay are: (a) the immobilization and/or patterning of target-specific bio-molecules on the surface of a microfluidic channel, for selectively capturing bio-targets like antigens or pathogens, followed by (b) sensitive detection of the bio-targets. In this thesis, we demonstrate microfluidic bio-assays based on novel methods for generating protein-patterns and on sensitive detection of the bio-targets. First, we introduce a simple and fast method for creating protein micropatterns both on a bare substrate and in-situ inside a microfluidic channel, in a matter of minutes, through electrostatic self-assembly of pre-functionalized magnetic beads. A lift-off patterned positively-charged aminosilane layer is used as the template for immobilizing the protein-coated negatively-charged beads. The number and arrangement of the beads can be well-controlled by altering the silane template design. Subsequently, we use patterned beads as assay substrates for performing on-chip bioassays. We demonstrate highly-sensitive full on-chip sandwich immunoassays for single and multi-analyte detection using beads as assay substrate. We successfully explored the possibility to lower the detection limit of immunoassays by concentrating the target antigens on a very small number of patterned beads. We also present the application of bead patterns as a platform for immuno-separation, culture and analysis of target (cancer) cells. Finally, we demonstrate a rapid on-chip immuno-histo-chemical assay on breast cancer tissues. We use luminescent lanthanide probes in place of conventional fluorescent probes, as labels for detection antibodies, for sensitive detection and quantification of biomarkers. Thanks to the time resolved microscopy and luminescent probes, the background noise due to the autofluorescence of the samples (i.e. tissue, cells) and microfluidic chips is successfully eliminated resulting in an improved signal-to-noise ratio when compared with the fluorescent microscopy results. Our assay results fully agree with the clinical analyses outcome, and this opens perspectives for a fully-integrated cancer detection platform for bedside diagnostics