Konstantinos Mitsakakis

Quantitative Determination of Size and Shape of Surface-Bound DNA Using an Acoustic Wave Sensor

DNA bending plays a significant role in many biological processes, such as gene regulation, DNA replication, and chromosomal packing. Understanding how such processes take place and how they can, in turn, be regulated by artificial agents for individual oriented therapies is of importance to both biology and medicine. In this work, we describe the application of an acoustic wave device for characterizing the conformation of DNA molecules tethered to the device surface via a biotin-neutravidin interaction. The acoustic energy dissipation per unit mass observed upon DNA binding is directly related to DNA intrinsic viscosity, providing quantitative information on the size and shape of the tethered molecules. The validity of the above approach was verified by showing that the predesigned geometries of model double-stranded and triple-helix DNA molecules could be quantitatively distinguished: the resolution of the acoustic measurements is sufficient to allow discrimination between same size DNA carrying a bent at different positions along the chain. Furthermore, the significance of this analysis to the study of biologically relevant systems is shown during the evaluation of DNA conformational change upon protein (histone) binding.

Multi-sample acoustic biosensing microsystem for protein interaction analysis

The current work describes a novel setup for multi-sample biomolecular analysis. It is based on the assembly of a dual acoustic device chip with a four-channel microfluidic module, forming an array of eight available domains for experiments. Initially, multiple detection was demonstrated via the specific interaction of neutravidin with four different biotinylated proteins, namely protein G, protein A, bovine serum albumin, and immunoglobulin G; results revealed a reproducibility between the microchannel domains better than 90%. Real-time analysis of the binding interactions was used to calculate the affinity and kinetic constants of the four biotinylated molecules binding to surface-immobilized neutravidin; this was the first time that this information was derived using a biosensing device and four biotinylated molecules. Interestingly, all calculated kinetic and affinity constants resemble those typical of antibody-antigen interactions, although the investigated specific binding was of avidin-biotin nature. Finally, under device pre-functionalization conditions, it was possible to probe eight interactions all together, exploiting the full capacity of the microsystem and reducing significantly the analysis time, contrary to the use of the standard acoustic device configuration. The outcome of this full-scale validation opens the way for the integrated acoustic platform to be implemented in even higher throughput detection for future diagnostic/biomedical applications, as well as in fundamental research studies regarding biomolecular interaction investigation and characterization.

Detection of multiple cardiac markers with an integrated acoustic platform for cardiovascular risk assessment.

This work describes the application of a newly emerged biosensing configuration incorporating a Surface Acoustic Wave device integrated with a multi-channel microfluidic module for the rapid and efficient analysis of cardiac markers. The examined cardiac markers of creatine kinase MB (CK-MB), cardiac reactive protein (CRP), D-dimer and pregnancy-associated plasma protein A (PAPP-A), comprise a group of both established and emerging heart disease proteins, that has never been probed before with any kind of biochip-related platform. The four markers were successfully detected; kinetics and affinity studies on their interactions with the surface immobilized antibodies are also presented. A concentration detection limit of less than 1 nM was achieved, with a dynamic range of more than two orders of magnitude, covering some of the pathological and healthy areas of interest. Mixtures of biomarkers applied to the device surface were used to prove the specificity of each binding event and investigate the microsystems performance in the presence of complex fluids, towards future utilization with real samples. The simplicity and multiplexing ability of the integrated platform render the system ideal as a potential diagnostic tool for cardiovascular risk assessment, where simultaneous analysis of various protein markers is required.

Integration of Microfluidics With a Love Wave Sensor for the Fabrication of a Multisample Analytical Microdevice

A novel approach for multisensing has been developed based on the integration of a parallel-channel microfluidic module with a surface acoustic wave (SAW) sensor chip. The microfluidic module was used to compartmentalize the surface of a single SAW sensor into equal subareas in order to deliver and detect multiple samples on the sensor. The design concerns and fabrication procedure using soft lithography of polydimethylsiloxane are described. Successful demonstration of a four-channel module is reported, along with a sensitivity evaluation and comparison with a standard flow cell used so far. Very promising results were revealed during the tests concerning the systems operation with liquid samples. The reliability and reproducibility of the results in all four subareas render the proposed setup very suitable for biological testing and screening of various biomolecules in an array format.

Characterization of DNA-Hv1 histone interactions; discrimination of DNA size and shape.

We have studied the formation of histone Hv1–DNA complexes using an acoustic biosensor and AFM imaging. Our results show that DNA and histone molecules aggregate into amorphous accumulations which form a compact rigid layer on the sensors surface. By measuring changes in the acoustic wave amplitude, it was possible to titrate surface bound DNA with Hv1 and discriminate between DNA molecules of different size and shape. From the kinetic analysis of real time data, K eq was found equal to 3 × 105 M−1.

Current problems associated with the microbiological point-of-care testing of respiratory tract infections in primary care

Department of Medical Microbiology & Infectious Diseases, Erasmus University Medical Center Rotterdam (Erasmus MC), Wytemaweg 80, 3015 CN, Rotterdam, The Netherlands Department of General Practice, Erasmus University Medical Center Rotterdam (Erasmus MC), Wytemaweg 80, 3015 CN, Rotterdam, The Netherlands Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany Laboratory for MEMS Applications, IMTEK – Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany *Author for correspondence: j.hays@erasmusmc.nl

Developmental roadmap for antimicrobial susceptibility testing systems

Antimicrobial susceptibility testing (AST) technologies help to accelerate the initiation of targeted antimicrobial therapy for patients with infections and could potentially extend the lifespan of current narrow-spectrum antimicrobials. Although conceptually new and rapid AST technologies have been described, including new phenotyping methods, digital imaging and genomic approaches, there is no single major, or broadly accepted, technological breakthrough that leads the field of rapid AST platform development. This might be owing to several barriers that prevent the timely development and implementation of novel and rapid AST platforms in health-care settings. In this Consensus Statement, we explore such barriers, which include the utility of new methods, the complex process of validating new technology against reference methods beyond the proof-of-concept phase, the legal and regulatory landscapes, costs, the uptake of new tools, reagent stability, optimization of target product profiles, difficulties conducting clinical trials and issues relating to quality and quality control, and present possible solutions.This Consensus Statement presents the barriers that currently prevent the timely development and implementation of novel and rapid antimicrobial susceptibility testing platforms, including the costs involved, uptake of new tools, legal and regulatory aspects, difficulties conducting clinical trials and quality control, and presents possible solutions.

Converging Human and Malaria Vector Diagnostics with Data Management towards an Integrated Holistic One Health Approach

Monitoring malaria prevalence in humans, as well as vector populations, for the presence of Plasmodium, is an integral component of effective malaria control, and eventually, elimination. In the field of human diagnostics, a major challenge is the ability to define, precisely, the causative agent of fever, thereby differentiating among several candidate (also non-malaria) febrile diseases. This requires genetic-based pathogen identification and multiplexed analysis, which, in combination, are hardly provided by the current gold standard diagnostic tools. In the field of vectors, an essential component of control programs is the detection of Plasmodium species within its mosquito vectors, particularly in the salivary glands, where the infective sporozoites reside. In addition, the identification of species composition and insecticide resistance alleles within vector populations is a primary task in routine monitoring activities, aiming to support control efforts. In this context, the use of converging diagnostics is highly desirable for providing comprehensive information, including differential fever diagnosis in humans, and mosquito species composition, infection status, and resistance to insecticides of vectors. Nevertheless, the two fields of human diagnostics and vector control are rarely combined, both at the diagnostic and at the data management end, resulting in fragmented data and mis- or non-communication between various stakeholders. To this direction, molecular technologies, their integration in automated platforms, and the co-assessment of data from multiple diagnostic sources through information and communication technologies are possible pathways towards a unified human vector approach.

An integrated microfluidics-on-SAW (“μF-on-SAW”) setup for multi-sample sensing

This work describes the design and fabrication of a novel approach to achieve multi-sample sensing based on a single surface acoustic wave (SAW) device. This is achieved with the integration of a multi-channel microfluidic module, which divides a SAW device into N discrete sub-areas, each of them capable of hosting an individual experiment. The fabrication of the micromodule was realized by means of soft lithography of poly(dimethylsiloxane) - PDMS. Evaluation of the setup showed successful operation with liquid samples. Regarding the sensing performance of the new setup, experiments proved that it exhibits remarkable reproducibility in the signal output and sensitivity among the sub-areas. The microfluidics-on-SAW setup, thus, appears to be a robust and reliable system for use in the cases that multi-analyte sensing is needed, such as biological applications regarding biomarker detection, drug screening, etc.