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The paper presents a new multi-parametric protein microarray embracing the multi-analyte capabilities of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The combination of high throughput reverse phase protein microarrays with element tagged antibodies and LA-ICP-MS makes it possible to detect and quantify many proteins or biomarkers in multiple samples simultaneously. A proof of concept experiment is performed for the analysis of cytochromes particularly of cytochrome P450 enzymes, which play an important role in the metabolism of xenobiotics such as toxicants and drugs. With the aid of the LA-ICP-MS based multi-parametric reverse phase protein microarray it was possible to analyse 8 cytochromes in 14 different proteomes in one run. The methodology shows excellent detection limits in the lower amol range and a very good linearity of R² ≥ 0.9996 which is a prerequisite for the development of further quantification strategies.
The importance of microRNA (miRNA) dysregulation for the development and progression of diseases and the discovery of stable miRNAs in peripheral blood have made these short-sequence nucleic acids next-generation biomarkers. Here we present a fully homogeneous multiplexed miRNA FRET assay that combines careful biophotonic design with various RNA hybridization and ligation steps. The single-step, single-temperature, and amplification-free assay provides a unique combination of performance parameters compared to state-of-the-art miRNA detection technologies. Precise multiplexed quantification of miRNA-20a, -20b, and -21 at concentrations between 0.05 and 0.5 nm in a single 150 mL sample and detection limits between 0.2 and 0.9 nm in 7.5 mL serum samples demonstrate the feasibility of both highthroughput and point-of-care clinical diagnostics.
In recent years, much progress has been made in analyzing the molecular origin of many diseases in vivo. For most applications, attention has been devoted to the detection of single molecules only. In this study, we present a proof of concept for the straightforward monitoring of interactions between different molecules via Förster resonance energy transfer (FRET) in an in vivo spectral multiplexing approach using conventional small organic dyes covalently attached to antibodies. Methods: We coupled the fluorophores DY-682 (donor; absorption [abs]/emission [em], 674/712 nm), DY-505 (control donor; abs/em, 498/529 nm), and DY-782 (acceptor; abs/em, 752/795 nm) to the model antibody IgG. The occurrence of FRET between these fluorophores was assessed in vitro for conjugate mixtures adsorbed onto membranes, after accumulation into the phagocytic compartment of macrophages (J774 cells), and in vivo in a mouse edema model using a whole-body animal imaging system with multispectral analysis features. Results: When the free acceptor DY-782 was combined with the DY-682 donor, FRET occurred as a consequence of small dye-to-dye distances, unlike the case for mixtures of the dyes DY-782 and DY-505. Our proof of concept was also transferred to living cells after internalization of the DY-682-IgGDY-782-IgG pair into macrophages and finally to animals, where intermolecular FRET was observed after systemic probe application in vivo in edema-bearing mice. Conclusion: Our simple cooperative-imaging approach enables the noninvasive detection of the presence of two or principally even more neighboring disease-related biomarkers. This finding is of high relevance for the in vivo identification of complex biologic processes requiring strong spatial interrelations of target molecules in key pathologic activation processes such as inflammation, cancer, and neurodegenerative diseases.
Luminescence-based detection methods, ranging from fluorescence spectroscopy for photophysical and mechanistic studies over sensing applications, chromatographic separation techniques and the microarray technology with fluorescence detection to fluorescence microscopy, flow cytometry, single molecule spectroscopy, and molecular imaging to integrating sphere spectroscopy, are among the most widely used methods in the life and material sciences. This is due to e.g., their unique sensitivity enabling the detection of single molecules, potential for multiplexing, ease of combination with spatial resolution, and suitability for remote sensing. Many of these advantages are closely linked to the choice of suitable molecular and nanoscale fluorescent reporters, typically required for signal generation. This includes organic dyes without and with sensor function, fluorophore-encoded polymeric and silica nanoparticles as well as nanocrystalline systems like semiconductor quantum dots and upconversion phosphors, emitting in the visible (vis), near-infrared (NIR), and IR (infrared). Current challenges present the environment sensitivity of most fluorophores, rendering fluorescence spectra, measured intensities/fluorescence quantum yields, and fluorescence decay kinetics matrix-dependent, and instrument-specific distortions of measured fluorescence signals that need to be considered for quantification and comparability of data, particularly fluorescence spectra.
Here, current applications of luminescence-based methods and different types of reporters will be presented. In this context, suitable spectroscopic tools for the characteri-zation of the optical properties of fluorescent reporters and fluorophore-encoded microparticles, analytical tools for the determination of the surface chemistry of different types of particles, and different multiplexing strategies will be discussed.
Analytical assays that can be used outside of a dedicated laboratory environment have received unprecedented attention all around the globe during the last one and a half years, in which the world was struggling with and trying to contain the SARS-CoV-2 pandemic. However, besides their current prominence in the field of medical diagnostics, rapid tests and assays have also become increasingly important in other areas ranging from food and feed via security and forensics to environmental management. The advantage is obvious: taking the assay to the sample instead of bringing the sample to the laboratory minimizes the time between first suspicion and first decision taking. Especially today, where mobile communication devices with powerful computing capabilities and onboard cameras are omnipresent, the majority of the global population possesses the basic skills of operating a powerful detector, ready at their fingertips. The stage is thus set for a much broader use of analytical measurements in terms of prognostics and prevention.
Today, however, tests for single parameters are still prevailing, whether it is a SARS-CoV-2 biomarker, the glucose level in blood or the concentration of lead in a water sample. Also, for industrial use, many mobile analytical systems still target a single parameter, and several separate runs or tests have to be used even if one wants to screen for only a small number of key parameters. Current challenges in the field thus lie with the development of low-number multiplexing strategies while allowing for robust, reliable, fast, and user-friendly operation and while still reaching the required sensitivities.
This lecture will give an overview of various generic approaches developed in BAM’s Chemical and Optical Sensing Division over the years to address these challenges. In particular, it will highlight how the combination of supramolecular (bio)chemistry, luminescence detection, hybrid (nano)materials and device miniaturization can result in powerful (bio)analytical assays that can be used at a point-of-need. Selected examples will introduce key aspects of such systems like tailored signaling mechanisms and recognition elements, materials functionalization and device integration, including hybrid mesoporous nanomaterials, gated indicator release systems, molecularly imprinted polymers, microfluidic devices, test strips and smartphone-based analysis.
An increasing number of (bio)analytical techniques rely on multiparametric analyses and the measurement of a very small number of emitters. While the former implies encoding or labeling by means of easily distinguishable properties like luminescence color or lifetime in conjunction with high-throughput optical-spectroscopic methods such as flow cytometry, the latter requires methods suitable for the characterization of the optical properties of single emitters. Here, we present the use of fluorescence correlation spectroscopy (FCS) for the relative determination of the key parameter photoluminescence quantum yield [5] and first results from flow cytometry measurements in the time-domain with a custom-designed instrument with luminescence lifetime analysis capability.
The simultaneous detection of different analytes has gained increasing importance in recent years, especially in the fields of medical diagnostics and environmental analysis. Multiplex assays allow for a range of biomarkers or pollutants to be rapidly and simultaneously measured. Particularly suitable formats for multiplexing are bead-based assays. The beads employed need to fulfil size and density requirements, important for instance for flow cytometry, and shall exhibit a high modularity to be facilely adapted to various kinds of analytes and detection systems. Core/shell particles are ideally suited in this sense because of their modularity in design and adaptability for various (bio)analytical assays. Here, polystyrene particles coated with different kinds of shells are presented, possessing features that are useful for a multitude of assay formats.
The particles in focus were coated with mesoporous and non-porous silica shells, with the possibility to introduce magnetic features to facilitate easier handling dependent on the assay format (e.g., in microfluidics). With high throughput analyses in mind, cytometric model assays were developed. Different factors such as pH or mediator salt used during shell preparation were evaluated with shell inspection by electron microscopy (SEM/TEM/EDX) being key to architectural control of the monodisperse particles. The optimized core/shell particles can be functionalized with capture biomolecules for toxins, viruses, or drugs to demonstrate particle performance. Showing how tailoring of the shell’s surface area controls sensitivity and dynamic range of the assay, an antibody-based assay for the detection of mycotoxins and a multiplex assay for the determination of DNA from different human papilloma virus (HPV) lines were developed.
Research of division Biophotonics at the Federal Institute for Materials Research and Testing (BAM) covers several topics including photophysics of molecular and nanocrystalline emitters, the development of signal enhancement, multiplexing, and barcoding strategies, surface group quantification, the rational design of different types of stimuli-responsive optical probes, and concepts and reference materials for the validation of optical-spectroscopic measurements. In the following representative examples for each of these topics are given. Also, current developments like single particle spectroscopy and flow cytometry with lifetime detection and newly certified fluorescence quantum yield standards are presented.
In particular, the rapid development of lateral flow assays as indispensable tools for everyone to contain the SARS-CoV-2 pandemic has fuelled the global demand for analytical tests that can be used outside dedicated laboratories. In addition to their use in medical diagnostics, rapid tests and assays have become increasingly important in various fields such as food safety, security, forensics, and environmental management. The advantage is obvious: taking the assay directly to the sample minimizes the time between suspicion and decision-making, allowing faster action. Especially today, when mobile communication devices with powerful computing capabilities and built-in cameras are ubiquitous, more people than ever before around the world have the basic skills to operate a powerful detector at their fingertips. This sets the stage for a much wider use of analytical measurements in terms of prognosis and prevention, enabling professional laypersons in particular.
However, current strip-based systems are primarily focused on single parameter analysis, whether it is SARS-CoV-2 biomarkers, blood glucose levels, or lead concentrations in water samples. Industrial applications of such methods also often still rely on single-parameter assays, requiring multiple runs even for a limited number of key parameters. Overcoming these limitations depends on developing low-number multiplexing strategies that ensure robustness, reliability, speed, ease of use, and sensitivity.
This lecture will give an overview of several generic approaches developed in recent years to address these challenges. It will highlight how the synergy of supramolecular (bio)chemistry, luminescence detection, hybrid (nano)materials and device miniaturization can result in powerful (bio)analytical assays that can be used at a point-of-need.1-5 Selected examples will introduce key aspects of such systems that include tailored signaling mechanisms and recognition elements, materials functionalization and device integration, including hybrid nanomaterials, gated indicator release systems, strip modification, and smartphone-based analysis.
The potential of semiconducting nanocrystals or so-called quantum dots (QDs) for lifetime multiplexing has not been investigated yet, despite the increasing use of QDs in (bio)analytical detection, biosensing, and fluorescence imaging and the obvious need for simple and cost-effective tools and strategies for the simultaneous detection of multiple analytes or events. This is most likely related to their multiexponential decay behavior as for multiplex chromophores, typically monoexponential decay kinetics are requested. The fluorescence decay kinetics of various mixtures of a long-lived, multiexponentially decaying CdSe QD and a short-lived organic dye were analyzed, and a model was developed for the quantification of these labels from the measured complex decay kinetics as a first proof-of-concept for the huge potential of these labels for lifetime multiplexing. In a second step, we evaluated the potential of mixtures of two types of QDs, varying in constituent material to realize distinguishable, yet multiexponential decay kinetics and similar absorption and emission spectra. Strategies for lifetime multiplexing with nanocrystalline labels were derived on the basis of these measurements.