TY - JOUR A1 - Morgner, Frank A1 - Stufler, Stefan A1 - Geissler, Daniel A1 - Medintz, Igor L. A1 - Algar, W. Russ A1 - Susumu, Kimihiro A1 - Stewart, Michael H. A1 - Blanco-Canosa, Juan B. A1 - Dawson, Philip E. A1 - Hildebrandt, Niko T1 - Terbium to quantum dot FRET Bioconjugates for clinical diagnostics influence of human plasma on optical and assembly properties JF - Sensors N2 - Forster resonance energy transfer (FRET) from luminescent terbium complexes (LTC) as donors to semiconductor quantum dots (QDs) as acceptors allows extraordinary large FRET efficiencies due to the long Forster distances afforded. Moreover, time-gated detection permits an efficient suppression of autofluorescent background leading to sub-picomolar detection limits even within multiplexed detection formats. These characteristics make FRET-systems with LTC and QDs excellent candidates for clinical diagnostics. So far, such proofs of principle for highly sensitive multiplexed biosensing have only been performed under optimized buffer conditions and interactions between real-life clinical media such as human serum or plasma and LTC-QD-FRET-systems have not yet been taken into account. Here we present an extensive spectroscopic analysis of absorption, excitation and emission spectra along with the luminescence decay times of both the single components as well as the assembled FRET-systems in TRIS-buffer, TRIS-buffer with 2% bovine serum albumin, and fresh human plasma. Moreover, we evaluated homogeneous LTC-QD FRET assays in QD conjugates assembled with either the well-known, specific biotin-streptavidin biological interaction or, alternatively, the metal-affinity coordination of histidine to zinc. In the case of conjugates assembled with biotin-streptavidin no significant interference with the optical and binding properties occurs whereas the histidine-zinc system appears to be affected by human plasma. KW - FRET KW - quantum dots KW - terbium KW - luminescence lifetime KW - blood KW - plasma KW - clinical diagnostics KW - biotin KW - streptavidin KW - histidin KW - immunoassay Y1 - 2011 U6 - https://doi.org/10.3390/s111009667 SN - 1424-8220 VL - 11 IS - 10 SP - 9667 EP - 9684 PB - MDPI CY - Basel ER - TY - JOUR A1 - Kupstat, Annette A1 - Kumke, Michael Uwe A1 - Hildebrandt, Niko T1 - Toward sensitive, quantitative point-of-care testing (POCT) of protein markers miniaturization of a homogeneous time-resolved fluoroimmunoassay for prostate-specific antigen detection JF - The analyst : the analytical journal of the Royal Society of Chemistry N2 - Point-of-care testing (POCT) systems which allow for a sensitive, quantitative detection of protein markers are extremely useful for the early detection and therapy progress monitoring of cancer. However, currently commercially available POCT devices are mainly limited to the qualitative detection of protein markers. In this study we demonstrate the successive miniaturization of a sensitive and fast assay for the quantitative detection of prostate-specific antigen (PSA) using a well established and clinically approved homogeneous time-resolved fluoroimmunoassay technology (TRACE (R)) on a commercial plate-reader system (KRYPTOR (R)). Regarding the initial requirements for the development of POCT devices we applied a 30-fold assay volume reduction (150 mu L to 5 mu L) to achieve a reasonable lab-on-a-chip volume and a 24-fold and 120-fold excitation pulse energy reduction to achieve reasonable pulse energies for low-cost miniature excitation sources. Due to highly efficient optimization of key POCT parameters our miniaturized PSA assay achieved a 30% increased sensitivity and a 2-fold improved limit of detection compared to the standard plate-reader method. Our results demonstrate the successful implementation of key parameters for a significant miniaturization and for cost reduction in the clinically approved KRYPTOR (R) platform for protein detection. The technological alterations required are easy-to-implement and can be immediately adapted for more than 30 diagnostic protein markers already available for the KRYPTOR (R) platform. These features strongly recommend our assay format to be utilized in innovative, sensitive, quantitative POCT of protein markers. Y1 - 2011 U6 - https://doi.org/10.1039/c0an00684j SN - 0003-2654 VL - 136 IS - 5 SP - 1029 EP - 1035 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Cywinski, Piotr J. A1 - Moro, Artur J. A1 - Ritschel, Thomas A1 - Hildebrandt, Niko A1 - Löhmannsröben, Hans-Gerd T1 - Sensitive and selective fluorescence detection of guanosine nucleotides by nanoparticles conjugated with a naphthyridine receptor JF - Analytical & bioanalytical chemistry N2 - Novel fluorescent nanosensors, based on a naphthyridine receptor, have been developed for the detection of guanosine nucleotides, and both their sensitivity and selectivity to various nucleotides were evaluated. The nanosensors were constructed from polystyrene nanoparticles functionalized by (N-(7-((3-aminophenyl) ethynyl)-1,8-naphthyridin- 2-yl) acetamide) via carbodiimide ester activation. We show that this naphthyridine nanosensor binds guanosine nucleotides preferentially over adenine, cytosine, and thymidine nucleotides. Upon interaction with nucleotides, the fluorescence of the nanosensor is gradually quenched yielding Stern-Volmer constants in the range of 2.1 to 35.9mM(-1). For all the studied quenchers, limits of detection (LOD) and tolerance levels for the nanosensors were also determined. The lowest (3 sigma) LOD was found for guanosine 3',5'-cyclic monophosphate (cGMP) and it was as low as 150 ng/ml. In addition, we demonstrated that the spatial arrangement of bound analytes on the nanosensors' surfaces is what is responsible for their selectivity to different guanosine nucleotides. We found a correlation between the changes of the fluorescence signal and the number of phosphate groups of a nucleotide. Results of molecular modeling and zeta-potential measurements confirm that the arrangement of analytes on the surface provides for the selectivity of the nanosensors. These fluorescent nanosensors have the potential to be applied in multi-analyte, array-based detection platforms, as well as in multiplexed microfluidic systems. KW - Naphthyridine receptor KW - cGMP KW - Base pairing KW - Nucleotide nanosensor KW - Fluorescence spectroscopy Y1 - 2011 U6 - https://doi.org/10.1007/s00216-010-4420-2 SN - 1618-2642 VL - 399 IS - 3 SP - 1215 EP - 1222 PB - Springer CY - Heidelberg ER -