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Arraying of single cells for mass spectrometric analysis is a considerable bioanalytical challenge. In this study, we employ a novel single cell arraying technology for quantitative analysis and isotopic fingerprinting by laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS). The single cell arraying approach is based on a piezo-acoustic microarrayer with software for automated optical detection of cells within the piezo dispense capillary (PDC) prior to arraying. Using optimized parameters, single cell occupancy of >99%, high throughput (up to 550 cells per hour), and a high cell recovery of >66% is achieved. LA-ICP-TOF-MS is employed to detect naturally occurring isotopes in the whole mass range as fingerprints of individual cells. Moreover, precise quantitative determination of metal-containing cell dyes is possible down to contents of ∼100 ag using calibration standards which were produced using the same arrayer.
Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput “all-on-one chip” system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide.
The development of fast and cheap highthroughput platforms for the detection of environmental contaminants is of particular importance to understand the human-related impact on the environment. The application of DNA-directed immobilization (DDI) of IgG molecules is
currently limited to the clinical diagnostics scenario, possibly because of the high costs of production of such addressable platforms. We here describe the efficient and specific hybridization of an antibody−oligonucleotide conjugate to a short 12-mer capture probe. The specific antibody used is a monoclonal antibody against caffeine, a stimulant and important anthropogenic marker. With this work, we hope to contribute to broadening the application potential of DDI to environmental markers in order to develop cheaper and more stable high-throughput screening platforms for standard Routine analysis of pollutants in a variety of complex matrices.
DNA origami nanostructures provide a platform where dye molecules can be arranged with nanoscale accuracy allowing to assemble multiple fluorophores without dye−dye aggregation. Aiming to develop a bright and sensitive ratiometric sensor system, we systematically studied the optical properties of nanoarrays of dyes built on DNA Origami platforms using a DNA template that provides a high versatility of label choice at minimum cost. The dyes are arranged at distances, at which they efficiently interact by Förster resonance energy transfer (FRET). To optimize array brightness, the FRET efficiencies between the donor fluorescein (FAM) and the acceptor cyanine 3 were determined for different sizes of the array and for different arrangements of the dye molecules within the array. By utilizing nanoarrays providing optimum FRET efficiency and brightness, we subsequently designed a ratiometric pH nanosensor using coumarin 343 as a pH-inert FRET donor and FAM as a pHresponsive acceptor. Our results indicate that the sensitivity of a ratiometric sensor can be improved simply by arranging the dyes into a well-defined array. The dyes used here can be easily replaced by other analyte-responsive dyes, demonstrating the huge potential of DNA nanotechnology for light harvesting, signal enhancement, and sensing schemes in life sciences.