Josef Horak

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.

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.

Amperometric micro-immunosensor for rapid Substance-P quantification in biological fluids

A capillary immunosensor-chip has been developed for fast determination of substance-p (SP) levels in biological fluids. The microdevice approach combines the advantages of the microfluidic capillary for implementation of a competitive immunoassay retaining sensitivity and specificity, with the highly sensitive electrochemical detection of GOD-tracer by flow-injection analysis. The assay exhibits a detection limit of 10 pg/mL. The immunosensor has been successfully used for SP quantification in blood plasma and saliva of healthy human volunteers: 110 pg/mL in plasma and 200 pg/mL in saliva were measured. The developed miniaturized immunoassay showed good precision, high sensitivity, acceptable stability and reproducibility with consistent results comparable to those obtained by the commercial assay kit. According to this, the proposed on-chip immunosensing method may have a potential applicability in medical diagnostics of major diseases.

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).

Amperometric Monitoring of Substance-P Levels in Biological Fluids

In this paper, a foil-based microfluidic chip (Fig. 1) for rapid determination of the neuropeptide Substance-P (SP) level in biological fluids is presented. Compared with standard ELISA methods, the miniaturization allows reducing the assay preparation and the measurement time. Due to the high variability levels and the extremely poor stability of SP in blood plasma, such improvement is crucial in point of care diagnostics of SP-induced inflammatory and immune diseases. The measurement is realized by a competitive immunoassay based on the sequenced immobilization of primary and secondary capture antibody into the capillary channel of the sensor (Fig. 2). Subsequent oxidation of hydrogen peroxide generated by the SP-labeled glucose oxidase mixed with the plasma sample resulted in a current signal inversely proportional to SP concentration. The measured 160 pg/ml in human sample (Fig. 3) was repeatable and reproducible by the corresponding optical ELISA kit on microtiter plates. Combined sensitive detection limit of 10 pg/ml and two minutes response demonstrates that this on-chip immunosensing technique is ideally suited for clinical application.