Can Dincer

Multiplexed Point-of-Care Testing – xPOCT

Multiplexed point-of-care testing (xPOCT), which is simultaneous on-site detection of different analytes from a single specimen, has recently gained increasing importance for clinical diagnostics, with emerging applications in resource-limited settings (such as in the developing world, in doctors’ offices, or directly at home). Nevertheless, only single-analyte approaches are typically considered as the major paradigm in many reviews of point-of-care testing. Here, we comprehensively review the present diagnostic systems and techniques for xPOCT applications. Different multiplexing technologies (e.g., bead- or array-based systems) are considered along with their detection methods (e.g., electrochemical or optical). We also address the unmet needs and challenges of xPOCT. Finally, we critically summarize the in-field applicability and the future perspectives of the presented approaches.

Sensitive, rapid and quantitative detection of substance P in serum samples using an integrated microfluidic immunochip.

Miniaturized diagnostic devices hold the promise of accelerate the specific and sensitive detection of various biomarkers, which can translate into many areas of medicine - from cheaper clinical trials, to early diagnosis and treatment of complex diseases. Therefore, we report on a disposable integrated chip-based capillary immunoassay featuring a microfluidic ELISA format combining electrochemical detection and low-cost fabrication employing a dry film photoresist, Vacrel(®) 8100. The readily accessible carboxylate groups on the material surface allow fast and high yield immobilization of biomolecules using amine-specific coupling via reactive esters requiring no laborious surface pretreatment. The integrated microfluidic system provides a convenient platform for a flow-through immunoassay. Capillary force is used for easy reagent delivery and loading the chip channel. We performed rapid quantification of serum level of substance P, a potential biomarker of acute neuroinflammation, using the developed microfluidic immunochip. Our miniaturized assay demonstrated a sensitive electrochemical detection of the antigen at 15.4pgml(-1) (11.5pM) using only 5µl of the biological fluid while cutting the total assay preparation time in half and the read-out time to 10min. Combining microfluidics and fabrication suitable for mass production with the capability of testing clinically relevant samples creates conditions for the construction of low-cost and portable point of care diagnostic devices with minimal auxiliary electronics.

Lift-Off Free Fabrication Approach for Periodic Structures with Tunable Nano Gaps for Interdigitated Electrode Arrays.

We report a simple, low-cost and lift-off free fabrication approach for periodic structures with adjustable nanometer gaps for interdigitated electrode arrays (IDAs). It combines an initial structure and two deposition process steps; first a dielectric layer is deposited, followed by a metal evaporation. The initial structure can be realized by lithography or any other structuring technique (e.g., nano imprint, hot embossing or injection molding). This method allows the fabrication of nanometer sized gaps and completely eliminates the need for a lift-off process. Different substrate materials like silicon, Pyrex or polymers can be used. The electrode gap is controlled primarily by sputter deposition of the initial structure, and thus, adjustable gaps in the nanometer range can be realized independently of the mask or stamp pattern. Electrochemical characterizations using redox cycling in ferrocenemethanol (FcMeOH) demonstrate signal amplification factors of more than 110 together with collection factors higher than 99%. Furthermore, the correlation between the gap width and the amplification factor was studied to obtain an electrochemical performance assessment of the nano gap electrodes. The results demonstrate an exponential relationship between amplification factor and gap width.

Designed miniaturization of microfluidic biosensor platforms using the stop-flow technique

Here, we present a novel approach to increase the degree of miniaturization as well as the sensitivity of biosensor platforms by the optimization of microfluidic stop-flow techniques independent of the applied detection technique (e.g. electrochemical or optical). The readout of the labeled bioassays, immobilized in a microfluidic channel, under stop-flow conditions leads to a rectangular shaped peak signal. Data evaluation using the peak height allows for a high level miniaturization of the channel geometries. To study the main advantages and limitations of this method by numerical simulations, a universally applicable model system is introduced for the first time. Consequently, proof-of-principle experiments were successfully performed with standard and miniaturized versions of an electrochemical biosensor platform utilizing a repressor protein-based assay for tetracycline antibiotics. Herein, the measured current peak heights are the same despite the sextuple reduction of the channel dimensions. Thus, this results in a 22-fold signal amplification compared to the constant flow measurements in the case of the miniaturized version.

Clinical on-site monitoring of ß-lactam antibiotics for a personalized antibiotherapy

An appropriate antibiotherapy is crucial for the safety and recovery of patients. Depending on the clinical conditions of patients, the required dose to effectively eradicate an infection may vary. An inadequate dosing not only reduces the efficacy of the antibiotic, but also promotes the emergence of antimicrobial resistances. Therefore, a personalized therapy is of great interest for improved patients’ outcome and will reduce in long-term the prevalence of multidrug-resistances. In this context, on-site monitoring of the antibiotic blood concentration is fundamental to facilitate an individual adjustment of the antibiotherapy. Herein, we present a bioinspired approach for the bedside monitoring of free accessible ß-lactam antibiotics, including penicillins (piperacillin) and cephalosporins (cefuroxime and cefazolin) in untreated plasma samples. The introduced system combines a disposable microfluidic chip with a naturally occurring penicillin-binding protein, resulting in a high-performance platform, capable of gauging very low antibiotic concentrations (less than 6 ng ml−1) from only 1 µl of serum. The system’s applicability to a personalized antibiotherapy was successfully demonstrated by monitoring the pharmacokinetics of patients, treated with ß-lactam antibiotics, undergoing surgery.

Self-assembled magnetic bead chains for sensitivity enhancement of microfluidic electrochemical biosensor platforms

In this paper, we present a novel approach to enhance the sensitivity of microfluidic biosensor platforms with self-assembled magnetic bead chains. An adjustable, more than 5-fold sensitivity enhancement is achieved by introducing a magnetic field gradient along a microfluidic channel by means of a soft-magnetic lattice with a 350 μm spacing. The alternating magnetic field induces the self-assembly of the magnetic beads in chains or clusters and thus improves the perfusion and active contact between the analyte and the beads. The soft-magnetic lattices can be applied independent of the channel geometry or chip material to any microfluidic biosensing platform. At the same time, the bead-based approach achieves chip reusability and shortened measurement times. The bead chain properties and the maximum flow velocity for bead retention were validated by optical microscopy in a glass capillary. The magnetic actuation system was successfully validated with a biotin-streptavidin model assay on a low-cost electrochemical microfluidic chip, fabricated by dry-film photoresist technology (DFR). Labelling with glucose oxidase (GOx) permits rapid electrochemical detection of enzymatically produced H2O2.

Biosensors and personalized drug therapy: what does the future hold?

In recent years, personalized medicine (PM), targeting at a tailored drug therapy or preventive care as individualized as the disease itself, is getting increasingly important in human medicine, pharmaceutical, and health-care industry. One of the important concepts of PM is ‘the right drug for the right patient at the right dose and time’ [1]. So, after the decision for a medication, a personalized drug therapy, which customizes the dose, dosage intervals, and the duration of the treatment to cover the patients’ individual needs, is vital. Nowadays, patients receive mostly the same standardized dose of a particular drug, independent of their clinical conditions. Although many different parameters, like health status (e.g. organ functions, infections, and genetic factors), metabolism (e.g. age, sex, and nutrition), or other physical factors (e.g. body weight), play a part in variable drug response, they are often not sufficiently taken into account. The first step toward an individualized drug therapy is therapeutic drug monitoring (TDM), the clinical measurement of medication in a human body fluid (e.g. blood, saliva, or urine) at certain time intervals during the treatment [2]. It aims to keep the drug concentration constantly over a certain threshold value, the so-called minimal inhibitory concentration, in the patient’s bloodstream, while personalizing the drug regimen. In this regard, the (quasi) real-timemonitoring of the pharmacokinetics, which describes all underlying processes (absorption, distribution, metabolism, and elimination) of a drug administered to a living organism, is crucial. Such an approach would be helpful mainly for drugs with narrow therapeutic windows, with known pharmacokinetic variability or with high risk for adverse effects (e.g. toxicity). These include, for example, antibiotics (e.g. aminoglycosides, vancomycin, or ß-lactams), antidepressants, antipsychotics, caffeine (in case of apnea in preterm infants), as well as immunosuppressive, antiarrhythmic (e.g. digoxin), and many antiepileptic agents for chronic therapy [3]. One of the main causes for the pharmacokinetic variability is the diverse renal function of patients as most of the drugs are eliminated by the kidneys. Therefore, the supplemental surveillance of kidney functionality during the pharmacotherapy could also be considered for the further improvement of the personalized drug treatment. The glomerular filtration rate (GFR) is a measure of the renal function. It is commonly gauged by the clearance measurements of exogenous biomarkers (e.g. inulin or iohexol) in urine samples. Yet, these tests are entailed with high running costs and long turnaround times, and therefore, cannot be integrated into clinical routine practice. Moreover, it is very problematic to ensure that the 24-hour urine sample is completely and properly collected by the patients themselves. For this reason, endogenous biomarkers, which are generated at a constant rate and eliminated only by kidneys, will be of great value for GFR testing. Herein, creatinine and cystatin C are considered as suitable candidates [4]. In addition to the TDM, another key aspect is the determination of the therapy duration by measuring predictive and monitoring biomarkers to improve the success and to reduce side effects of the treatment. Especially for the anti-infective therapy (e.g. in sepsis or other infections), near-patient surveillance of the progress of bacterial infections would provide valuable information for PM. In this sense, various biomarkers associated with bacterial inflammation (e.g. cytokines like interleukin-6, or c-reactive protein or procalcitonin) are of great interest [5]. Even the quasi real-time measurement of such biomarkers, indicating the state and progression of the infection, will have a major impact on the patient’s outcome. Additionally, it will allow for the identification of the clinical end point of the disease and thus, the determination of the treatment duration. To meet the needs and challenges of the personalized drug therapy appropriately, the multiplexed on-site monitoring of various substances over time, covering TDM or other therapyrelated issues like renal function or infection status, would be highly desirable. In this sense, biosensors are considered as powerful analytical tools. Since their discovery by Leland C. Clark, Jr. in 1962, biosensors have revolutionized not only the field of health care, but also food and environmental monitoring and so, have greatly improved the quality of our life. Nowadays, pregnancy tests and blood glucose meters are known by everyone since they allow for rapid and easy diagnosis or monitoring, performed at home even by the patients themselves. But how does a biosensor theoretically work? This compact analytical device incorporates a high-affinity recognition element along with a physicochemical transducer. The bioreceptor recognizes a (bio-)chemical event and converts this information into a measurable signal, which is gauged

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout. The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

Signal amplification using magnetic bead chains in microfluidic electrochemical biosensors

We present a novel approach to increase the sensitivity of microfluidic biosensor platforms using magnetic micro-bead chains. An almost 2-fold sensitivity enhancement is achieved by introducing a magnetic field gradient along a microfluidic channel by means of a soft-magnetic lattice with lattice spacings down to 100 μm. The magnetic field gradient induces self-assembly of the magnetic beads in chains or clusters and thus improves the active contact between analyte and beads. This facile strategy significantly increases the active bead surface while allowing for complete independence of traditional biosensor materials and channel geometries, chip-reusability and shortened measurement times. Bead chain properties were validated with optical microscopy in a glass capillary and with electrochemical measurements via glucose oxidase (GOx) labels on an integrated microfluidic chip fabricated in dry-film photo resist technology (DFR).