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3. Rapid parallel generation of a fluorescently barcoded drop library from a microtiter plate using the plate-interfacing parallel encapsulation (PIPE) chip.

Author

Zath, Geoffrey, Sperling, Ralph, Hoffman, Carter, Bikos, Dimitri, Abbasi, Reha, Weitz, David, Chang, Connie, and Abate, Adam

Subjects
Gene Library, Oligonucleotide Array Sequence Analysis, Microfluidics, Lab-On-A-Chip Devices, High-Throughput Screening Assays, Microfluidic Analytical Techniques
Abstract

In drop-based microfluidics, an aqueous sample is partitioned into drops using individual pump sources that drive water and oil into a drop-making device. Parallelization of drop-making devices is necessary to achieve high-throughput screening of multiple experimental conditions, especially in time-sensitive studies. Here, we present the plate-interfacing parallel encapsulation (PIPE) chip, a microfluidic chip designed to generate 50 to 90 μm diameter drops of up to 96 different conditions in parallel by interfacing individual drop makers with a standard 384-well microtiter plate. The PIPE chip is used to generate two types of optically barcoded drop libraries consisting of two-color fluorescent particle combinations: a library of 24 microbead barcodes and a library of 192 quantum dot barcodes. Barcoded combinations in the drop libraries are rapidly measured within a microfluidic device using fluorescence detection and distinct barcoded populations in the fluorescence drop data are identified using DBSCAN data clustering. Signal analysis reveals that particle size defines the source of dominant noise present in the fluorescence intensity distributions of the barcoded drop populations, arising from Poisson loading for microbeads and shot noise for quantum dots. A barcoded population from a drop library is isolated using fluorescence-activated drop sorting, enabling downstream analysis of drop contents. The PIPE chip can improve multiplexed high-throughput assays by enabling simultaneous encapsulation of barcoded samples stored in a microtiter plate and reducing sample preparation time.

Published
2022

7. DNA sequence analysis with droplet-based microfluidics

Author

Abate, Adam R, Hung, Tony, Sperling, Ralph A, Mary, Pascaline, Rotem, Assaf, Agresti, Jeremy J, Weiner, Michael A, and Weitz, David A

Subjects
Engineering, Chemical Sciences, Genetics, Biotechnology, Bioengineering, Generic health relevance, Base Sequence, High-Throughput Nucleotide Sequencing, Microfluidic Analytical Techniques, Sequence Analysis, DNA, Analytical Chemistry, Chemical sciences
Abstract

Droplet-based microfluidic techniques can form and process micrometer scale droplets at thousands per second. Each droplet can house an individual biochemical reaction, allowing millions of reactions to be performed in minutes with small amounts of total reagent. This versatile approach has been used for engineering enzymes, quantifying concentrations of DNA in solution, and screening protein crystallization conditions. Here, we use it to read the sequences of DNA molecules with a FRET-based assay. Using probes of different sequences, we interrogate a target DNA molecule for polymorphisms. With a larger probe set, additional polymorphisms can be interrogated as well as targets of arbitrary sequence.

Published
2013

14. Synchronized reinjection and coalescence of droplets in microfluidics

Author

Lee, Manhee, Collins, Jesse W., Aubrecht, Donald M., Sperling, Ralph, Solomon, Laura, Ha, Jong-Wook, Yi, Gi-Ra, Weitz, David A., and Publica

Abstract

Coalescence of two kinds of pre-processed droplets is necessary to perform chemical and biological assays in droplet-based microfluidics. However, a robust technique to accomplish this does not exist. Here we present a microfluidic device to synchronize the reinjection of two different kinds of droplets and coalesce them, using hydrostatic pressure in conjunction with a conventional syringe pump. We use a device consisting of two opposing T-junctions for reinjecting two kinds of droplets and control the flows of the droplets by applying gravity-driven hydrostatic pressure. The hydrostatic-pressure operation facilitates balancing the droplet reinjection rates and allows us to synchronize the reinjection. Furthermore, we present a simple but robust module to coalesce two droplets that sequentially come into the module, regardless of their arrival times. These re-injection and coalescence techniques might be used in lab-on-chip applications requiring droplets with controlled numbers of solid materials, which can be made by coalescing two pre-processed droplets that are formed and sorted in devices.

Published
2014

17. Surface Modification and Functionalization of Colloidal Nanoparticles

Author

Sperling, Ralph Alexander and Parak, Wolfgang (Prof.)

Subjects
Oberflächenchemie, Monodisperses System, Biokonjugation, Nichtwässriges Kolloid, Kolloidchemie, Nanopartikel, Kolloid, Naturwissenschaften, Nanocrystals, Quantenpunkt, Kolloidphysik, Nanoparticles, Colloids, Teilchen, Bionjugation, Functionalization, Nanokristalle, 2008, Sciences, ddc:500, Sciences -- Naturwissenschaften
Abstract

Subject of this cumulative dissertation is the surface modification, functionalization and bioconjugation of inorganic colloidal nanoparticles. Such particles have a diameter of about 1 – 100 nm and consist of an inorganic, crystalline core that is commonly covered by a shell of organic ligand molecules. These ligand molecules control the growth of the particles during synthesis and also stabilize the particles in solution against aggregation. Besides the core material, the particle surface determines the properties of the particles, as to their interaction with the surrounding medium and to chemical and biological functionality that can be modified by reactive groups and biomolecules. This work aimed to functionalize the surface of colloidal nanoparticles in a controlled fashion and to modify the particles with molecules that provide stability in biological relevant, aqueous environment and that possibly also provide specific interaction with biological systems. First hydrophobic nanoparticles, e.g. made of gold or CdSe/ZnS, were transferred from organic to the aqueous phase by means of an amphiphilic polymer. The polymer consists of carboxylic groups that stabilize the particles by electrostatic repulsion and provide anchor groups for further chemical functionalization. Besides the direct functionalization of the amphiphilic polymer in organic solution, different molecules could be bound to the polymer shell in aqueous solution, regardless to the core material. By means of poly(ethylene glycol), PEG, an inert linear polymer, the particles could be sterically stabilized. Bifunctional PEG with different functional groups allows for modification of the particles with new chemical functions. By means of sufficiently large PEG molecules, a separation of particles with exactly one or two reactive functional groups could be achieved by gel electrophoresis. For the size characterization of nanoparticles, different complementary methods were compared. The system was applied to particles of different core materials and different functional molecules. Complementary to this, citrate-stabilized gold nanoparticles were modified by thiol-modified DNA and characterized by gel electrophoresis which provided information about the DNA conformation. In a collaborative work it was demonstrated that such DNA-modified particles can be used for the detection of DNA oligomers by laser-induced melting of nanoparticle aggregates., Thema dieser kumulativen Dissertation ist die Oberflächenmodifikation, Funktionalisierung und Biokonjugation anorganischer kolloidaler Nanopartikel. Solche Partikel besitzen einen Durchmesser zwischen ca. 1 und 100 nm und bestehen aus einem anorganischen, kristallinen Kern, der üblicherweise durch eine Schicht organischer Ligandenmoleküle bedeckt ist. Diese Ligandenmoleküle kontrollieren einerseits das Wachstum der Nanokristalle bei der Synthese und stabilisieren andererseits die Partikel in Lösung gegen Aggregation. Neben dem Kernmaterial bestimmt die Oberfläche maßgeblich die Eigenschaften der Nanopartikel, in Hinsicht auf das Verhalten im umgebenden Medium sowie die chemische und biologische Funktionalität, die durch chemische Gruppen und Biomoleküle modifiziert werden kann. Ziel dieser Arbeit war es, die Oberfläche von kolloidalen Nanopartikeln kontrolliert zu funktionalisieren und mit Molekülen zu versehen, die die Partikel in biologisch relevanter, wässriger Umgebung stabil halten und ggf. eine spezifische Wechselwirkung mit biologischen Systemen ermöglichen. Zunächst wurden hydrophobe Nanopartikel aus z.B. Gold oder CdSe/ZnS mit Hilfe eines amphiphilen Polymers von der organischen in die wässrige Phase überführt. Das Polymer besitzt Carboxylgruppen, die die Partikel durch elektrostatische Abstoßung stabilisieren und als Ankergruppen für die weitere chemische Funktionalisierung dienen. Neben der direkten Funktionaliserung des amphiphilen Polymers in organischer Lösung konnten verschiedene Moleküle an die Nanopartikel mit Polymerhülle in wässriger Lösung angebunden werden, unabhängig vom Kernmaterial der Partikel. Mit Hilfe von Polyethylenglycol (PEG), einem inerten linearen Polymer, konnten die Partikel zusätzlich sterisch stabilisiert werden. Ein bifunktionales PEG mit verschiedenen funktionellen Endgruppen erlaubte es, die Partikel mit neuen chemischen Funktionen zu versehen. Bei hinreichend großen PEG Molekülen konnte durch Gelelektrophorese eine Trennung von Partikeln mit genau einer oder zwei reaktiven funktionellen Gruppen erreicht werden. Für die Größencharakterisierung von Nanopartikeln wurden verschiedene komplementäre Methoden miteinander verglichen. Das System wurde auf verschiedene Kernmaterialien und funktionale Moleküle erweitert. Komplementär dazu wurden citrat-stabilisierte Goldpartikel mit thiol-modifizierter DNA versehen und durch Gel-Elektrophorese charakterisiert, was über den Partikeldurchmesser Rückschlüsse auf die DNA Konformation ermöglichte. Zusätzlich wurde im Rahmen einer Kollaboration gezeigt, dass solche DNA-modifizierte Partikel dazu verwendet werden können, DNA-Oligomere durch Laser-induziertes Aufschmelzen von Partikelaggregaten nachzuweisen.

Published
2009
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18. One-Pot Synthesis of Cationic Gold Nanoparticles by Differential Reduction.

Author

Sperling, Ralph A., García-Fernández, Lorena, Ojea-Jiménez, Isaac, Piella, Jordi, Bastús, Neus G., and Puntes, Víctor

Subjects
GOLD nanoparticle synthesis, SURFACE active agents, NANOSTRUCTURED materials
Abstract

The size-controlled synthesis of cationic particles by differential reduction of HAuCl4 precursor in the presence of NaBH4 and 1-aminoundecane-12-thiol (AUT) is reported. The number of seed particles is determined by the fraction of the initially Au precursor reduced by NaBH4 present in the reaction mixture, which are then grown larger by the AUT, acting as both weak reducing agent and stabilizing surfactant. By this methodology, size controlled synthesis is achieved in a two-step one-pot synthesis at room temperature. [ABSTRACT FROM AUTHOR]

Published
2017
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21. Air–Blood Barrier Translocation of Tracheally Instilled Gold Nanoparticles Inversely Depends on Particle Size

Author

Kreyling, Wolfgang G., primary, Hirn, Stephanie, additional, Möller, Winfried, additional, Schleh, Carsten, additional, Wenk, Alexander, additional, Celik, Gülnaz, additional, Lipka, Jens, additional, Schäffler, Martin, additional, Haberl, Nadine, additional, Johnston, Blair D., additional, Sperling, Ralph, additional, Schmid, Günter, additional, Simon, Ulrich, additional, Parak, Wolfgang J., additional, and Semmler-Behnke, Manuela, additional

Published
2013
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29. DNA Melting in Gold Nanostove Clusters

Author

Hrelescu, Calin, primary, Stehr, Joachim, additional, Ringler, Moritz, additional, Sperling, Ralph A., additional, Parak, Wolfgang J., additional, Klar, Thomas A., additional, and Feldmann, Jochen, additional

Published
2010
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35. Synthesis, Characterization, and Bioconjugation of Fluorescent Gold Nanoclusters toward Biological Labeling Applications

Author

Lin, Cheng-An J., primary, Yang, Ting-Ya, additional, Lee, Chih-Hsien, additional, Huang, Sherry H., additional, Sperling, Ralph A., additional, Zanella, Marco, additional, Li, Jimmy K., additional, Shen, Ji-Lin, additional, Wang, Hsueh-Hsiao, additional, Yeh, Hung-I, additional, Parak, Wolfgang J., additional, and Chang, Walter H., additional

Published
2009
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38. Gold NanoStoves for Microsecond DNA Melting Analysis

Author

Stehr, Joachim, primary, Hrelescu, Calin, additional, Sperling, Ralph A., additional, Raschke, Gunnar, additional, Wunderlich, Michael, additional, Nichtl, Alfons, additional, Heindl, Dieter, additional, Kürzinger, Konrad, additional, Parak, Wolfgang J., additional, Klar, Thomas A., additional, and Feldmann, Jochen, additional

Published
2008
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40. Synthesis and Characterization of Polymer-Coated Quantum Dots with Integrated Acceptor Dyes as FRET-Based Nanoprobes

Author

Fernández-Argüelles, María Teresa, primary, Yakovlev, Aleksey, additional, Sperling, Ralph A., additional, Luccardini, Camilla, additional, Gaillard, Stéphane, additional, Sanz Medel, Alfredo, additional, Mallet, Jean-Maurice, additional, Brochon, Jean-Claude, additional, Feltz, Anne, additional, Oheim, Martin, additional, and Parak, Wolfgang J., additional

Published
2007
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41. Size and Surface Effects on the MRI Relaxivity of Manganese Ferrite Nanoparticle Contrast Agents

Author

Tromsdorf, Ulrich I., primary, Bigall, Nadja C., additional, Kaul, Michael G., additional, Bruns, Oliver T., additional, Nikolic, Marija S., additional, Mollwitz, Birgit, additional, Sperling, Ralph A., additional, Reimer, Rudolph, additional, Hohenberg, Heinz, additional, Parak, Wolfgang J., additional, Förster, Stephan, additional, Beisiegel, Ulrike, additional, Adam, Gerhard, additional, and Weller, Horst, additional

Published
2007
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44. Quantifying cell-generated mechanical forces within living embryonic tissues.

Author

Campàs, Otger, Mammoto, Tadanori, Hasso, Sean, Sperling, Ralph A, O'Connell, Daniel, Bischof, Ashley G, Maas, Richard, Weitz, David A, Mahadevan, L, and Ingber, Donald E

Subjects
FETAL tissues, MORPHOGENESIS, PHYSIOLOGICAL stress, MICRODROPLETS, BIOMECHANICS, FLUORESCENCE microscopy
Abstract

Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. Little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing three-dimensional (3D) tissues in vivo. We present a method to quantify cell-generated mechanical stresses exerted locally within living embryonic tissues, using fluorescent, cell-sized oil microdroplets with defined mechanical properties and coated with adhesion receptor ligands. After a droplet is introduced between cells in a tissue, local stresses are determined from droplet shape deformations, measured using fluorescence microscopy and computerized image analysis. Using this method, we quantified the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates, and we confirmed that these stresses (3.4 nN μm−2) are dependent on myosin II activity and are more than twofold larger than stresses generated by cells of embryonic tooth mesenchyme, either within cultured aggregates or in developing whole mouse mandibles. [ABSTRACT FROM AUTHOR]

Published
2014
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46. Air–Blood Barrier Translocation of Tracheally Instilled Gold Nanoparticles Inversely Depends on Particle Size

Author

Kreyling, Wolfgang G., Hirn, Stephanie, Möller, Winfried, Schleh, Carsten, Wenk, Alexander, Celik, Gülnaz, Lipka, Jens, Schäffler, Martin, Haberl, Nadine, Johnston, Blair D., Sperling, Ralph, Schmid, Günter, Simon, Ulrich, Parak, Wolfgang J., and Semmler-Behnke, Manuela

Abstract

Gold nanoparticles (AuNP) provide many opportunities in imaging, diagnostics, and therapy in nanomedicine. For the assessment of AuNP biokinetics, we intratracheally instilled into rats a suite of 198Au-radio-labeled monodisperse, well-characterized, negatively charged AuNP of five different sizes (1.4, 2.8, 5, 18, 80, 200 nm) and 2.8 nm AuNP with positive surface charges. At 1, 3, and 24 h, the biodistribution of the AuNP was quantitatively measured by gamma-spectrometry to be used for comprehensive risk assessment. Our study shows that as AuNP get smaller, they are more likely to cross the air–blood barrier (ABB) depending strongly on the inverse diameter d–1of their gold core, i.e., their specific surface area (SSA). So, 1.4 nm AuNP (highest SSA) translocated most, while 80 nm AuNP (lowest SSA) translocated least, but 200 nm particles did not follow the d–1relation translocating significantly higher than 80 nm AuNP. However, relative to the AuNP that had crossed the ABB, their retention in most of the secondary organs and tissues was SSA-independent. Only renal filtration, retention in blood, and excretion viaurine further declined with d–1of AuNP core. Translocation of 5, 18, and 80 nm AuNP is virtually complete after 1 h, while 1.4 nm AuNP continue to translocate until 3 h. Translocation of negatively charged 2.8 nm AuNP was significantly higher than for positively charged 2.8 nm AuNP. Our study shows that translocation across the ABB and accumulation and retention in secondary organs and tissues are two distinct processes, both depending specifically on particle characteristics such as SSA and surface charge.

Published
2014
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47. One-Pot Synthesis of Cationic Gold Nanoparticles by Differential Reduction

Author

Sperling, Ralph A., García-Fernández, Lorena, Ojea-Jiménez, Isaac, Piella, Jordi, Bastús, Neus G., and Puntes, Víctor

Abstract

The size-controlled synthesis of cationic particles by differential reduction of HAuCl4precursor in the presence of NaBH4and 1-aminoundecane-12-thiol (AUT) is reported. The number of seed particles is determined by the fraction of the initially Au precursor reduced by NaBH4present in the reaction mixture, which are then grown larger by the AUT, acting as both weak reducing agent and stabilizing surfactant. By this methodology, size controlled synthesis is achieved in a two-step one-pot synthesis at room temperature.

Published
1966
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48. Corrigendum: Quantifying cell-generated mechanical forces within living embryonic tissues.

Author

Campàs, Otger, Mammoto, Tadanori, Hasso, Sean, Sperling, Ralph A, O'Connell, Daniel, Bischof, Ashley G, Maas, Richard, Weitz, David A, Mahadevan, L, and Ingber, Donald E

Subjects
CYTOLOGICAL research, EMBRYONIC stem cells
Abstract

A correction to the article related to quantifying cell-generated mechanical forces in living embryonic tissues is presented.

Published
2014
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49. Nitrogen-vacancy center magnetic imaging of Fe 3 O 4 nanoparticles inside the gastrointestinal tract of Drosophila melanogaster .

Author

Mathes N, Comas M, Bleul R, Everaert K, Hermle T, Wiekhorst F, Knittel P, Sperling RA, and Vidal X

Abstract

Widefield magnetometry based on nitrogen-vacancy centers enables high spatial resolution imaging of magnetic field distributions without a need for spatial scanning. In this work, we show nitrogen-vacancy center magnetic imaging of Fe 3 O 4 nanoparticles within the gastrointestinal tract of Drosophila melanogaster larvae. Vector magnetic field imaging based on optically detected magnetic resonance is carried out on dissected larvae intestine organs containing accumulations of externally loaded magnetic nanoparticles. The distribution of the magnetic nanoparticles within the tissue can be clearly deduced from the magnetic stray field measurements. Spatially resolved magnetic imaging requires the nitrogen-vacancy centers to be very close to the sample making the technique particularly interesting for thin tissue samples. This study is a proof of principle showing the capability of nitrogen-vacancy center magnetometry as a technique to detect magnetic nanoparticle distributions in Drosophila melanogaster larvae that can be extended to other biological systems., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Published
2023
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50. Chloroform- and water-soluble sol-gel derived Eu+++/Y2O3 (red) and Tb+++/Y2O3 (green) nanophosphors: synthesis, characterization, and surface modification.

Author

Pandey A, Roy MK, Pandey A, Zanella M, Sperling RA, Parak WJ, Samaddar AB, and Verma HC

Subjects
Color, Crystallization methods, Macromolecular Substances chemistry, Materials Testing, Molecular Conformation, Nanoparticles ultrastructure, Particle Size, Phase Transition, Solubility, Surface Properties, Chloroform chemistry, Europium chemistry, Luminescent Measurements methods, Nanoparticles chemistry, Nanotechnology methods, Terbium chemistry, Water chemistry
Abstract

Eu+++ and Tb+++ ions have been incorporated into nanodimensional yttrium oxide host matrices via a sol-gel process using Y5O(OPr(i))13 as precursor (OPr(i) = isopropoxy). The as-synthesized white powders have been annealed at different temperatures. Photoluminescence (PL) spectroscopy and X-ray diffraction (XRD) have been used as tools for documenting the characteristics of these powders. For Eu+++-doped powders, a comparison of the Eu+++, 5D0-->7F1, and 5D0-->7F2 peak intensities in the emission spectra reveals that the dopant ions are occupying unsymmetrical sites in the host yttrium oxide in all the samples. For Tb+++-doped powders, the characteristic terbium 5D3-->7Fn and 5D-->7Fn (n = 2-6) transitions were visible only in the samples that had been annealed above 500 degrees C. Samples of the doped particle powders were suspended in chloroform by fragmenting the powder with and without sonification under the presence of trioctylphosphine oxide, or a mixture of oleic acid and dioctyl ether. The resulting clear colorless (for Eu+++) and light green translucent (for Tb+++) solutions of the suspended particles showed red and green luminescence upon UV excitation, respectively. In addition, suspension in water has been achieved by fragmenting the powder in the presence of dichloroacetic acid. Transmission electron micrograph investigation of the soluble particles shows single dispersed particles along with agglomerates. The changes in the luminescence due to fragmentation of the particle powder and due the influence of the surfactant of the suspended colloidal particles are discussed.

Published
2009
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