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Quality control requirements imposed on assays used in clinical diagnostics and point-of-care-diagnostic testing (POCT), utilizing amplification reactions performed at elevated temperatures of 35 to 95 °C are very stringent. As the temperature of a reaction vessel has a large impact on the specificity and sensitivity of the amplification reaction, simple tools for local in situ temperature sensing and monitoring are required for reaction and assay control. We describe here a platform of stem-and-loop structured DNA hairpins (molecular beacons, MBs), absorbing and emitting in the visible and red spectral region, rationally designed for precise temperature measurements in microfluidic assays for POCT, and their ap-plication for temperature measurements in a common DNA-based molecular biological assay utilizing thermophilic helicase-dependent amplification (tHDA). Spectroscopic studies of these MBs, rationally designed from DNA se-quences of different thermal stabilities, chosen not to interact with the DNA probes applied in the nucleic acid amplification assay, and temperature-dependent fluorescence measurements of MB-assay mixtures revealed the suitability of these MBs for temperature measurements directly in such an assay with a temperature resolution of about 0.5 °C without interferences from assay components. Combining two spectrally distinguishable MBs provides a broader response range and an increase in temperature sensitivity up to 0.1 °C. This approach will find future application for temperature monitoring and quality control in commercialized diagnostics assays using dried reagents and microfluidic chips as well as assays read out with tube and microplate readers and PCR detection systems for temperature measurements in the range of 35 to 95 °C.
In Part II of this Review series on lanthanide-doped photon-upconversion nanoparticles (UCNPs), we present and critically discuss the Performance and suitability of UCNPs as background-free luminescent Reporters in bioimaging and bioanalytical applications. The preparation of a biocompatible nanoparticle surface is an integral step for all life - science-related applications. UCNPs have found their way into a large number of diagnostic platforms, homogeneous and heterogeneous assay formats, and sensor applications. Many bioanalytical detection schemes involve Förster resonance energy transfert (FRET), which is still debated for UCNPs and Needs to be much improved. The Need for dedicated and standardized instruments as well as recent studies on the Dissolution and potential toxicity of UCNPs are addressed. Finally we outline future Trends and challenges in the field of upconversion.
To demonstrate the potential of time-resolved flow cytometry (FCM) for bioanalysis, clinical diagnostics, and optically encoded bead-based assays, we performed a proof-of-principle study to detect biomolecular interactions utilizing fluorescence lifetime (LT)-encoded micron-sized polymer beads bearing target-specific bioligands and a recently developed prototype lifetime flow cytometer (LT-FCM setup). This instrument is equipped with a single excitation light source and different fluorescence detectors, one operated in the photon-counting mode for time-resolved measurements of fluorescence decays and three detectors for conventional intensity measurements in different spectral windows. First, discrimination of bead-bound biomolecules was demonstrated in the time domain exemplarily for two targets, Streptavidin (SAv) and the tumor marker human chorionic gonadotropin (HCG). In a second step, the determination of biomolecule concentration levels was addressed representatively for the inflammation-related biomarker tumor necrosis factor (TNF-α) utilizing fluorescence intensity measurements in a second channel of the LT-FCM instrument. Our results underline the applicability of LT-FCM in the time domain for measurements of biomolecular interactions in suspension assays. In the future, the combination of spectral and LT encoding and multiplexing and the expansion of the time scale from the lower nanosecond range to the longer nanosecond and the microsecond region is expected to provide many distinguishable codes. This enables an increasing degree of multiplexing which could be attractive for high throughput screening applications.
Biodetection formats, such as DNA and antibody microarrays, are valuable tools in the life sciences, but for some applications, the detection limits are insufficient. A straightforward strategy to obtain signal amplification is the rolling circle amplification (RCA), an easy, isothermal, and enzymatic nucleic acid synthesis that has already been employed successfully to increase the signal yield for several single-analyte and multiplexing assays in conjunction with hybridization probes. Here, we systematically investigated the parameters responsible for the RCA driven signal amplification with fluorescent labels, such as the type of fluorophore chosen, labeling strategy, composition of reaction solution, and number of handling steps. In labeling strategies, post-synthetic labeling via a Cy3-hybridization probe was compared to the direct incorporation of fluorescent Cy3–dUTP and DY-555–dUTP into the nascent strand during synthesis. With our direct labeling protocol, the assay's runtime and handling steps could be reduced while the signal yield was increased. These features are very attractive for many detection formats but especially for point-of-care diagnostic kits that need to be simple enough to be performed by scientifically untrained personnel.
In this review we present new concepts and recent progress in the application of semiconductur quantum dots (QD) as labels in two important areas of biology, bioimaging and biosensing. We analyze the biologically relevant properties of QDs focusing on the following topics: QD surface treatment and stability labeling of cellular structures and receptors with QDs, incorporation of QDs in living cells, cytotoxicity of QDs and influence of the biolocical environment on the biological and optical properties of QDs. Initially, we consider utilization of QDs as agants in high-resolution bioimaging techniques that can provide information at the molecular levels. The deverse range of modern live-cell QD-based imaging techniques with resolution far beyond the diffraction limit of light is examined. In each technique, we discuss the pros and cons of QD use and deliberate how QDs can be further engineered to facilitate their application in the respective imaging techniques and to produce significant improvements in resolution. Then we review QD-based point-of-care bioassays, bioprobes, and biosensors designed in different formats ranging from analytic biochemistry assays and ELISA, to novel point-of-care smartphone integrated QD-based biotests. Here, a wide range of QD-based fluorescence bioassays with optical transduction, electrochemiluminescence and photoelectrochemical assays are discussedc. Finally, this review provides an analysis of the prospects of application of QDs in selected important Areas of biology.
Immunoassays are suitable tools for high-throughput screenings. The prerequisite for accurate determinations by these methods is the selection of an excellent antibody. The production and selection of monoclonal antibodies is usually a tedious process. In this study, new strategies for improving antibody production and characterization were applied. This includes the monitoring of the immunization progress in mice through antibodies extracted from feces, which allows a time-resolved and animal-friendly monitoring of the immune response. Additionally, fluorescence polarization immunoassay (FPIA) could be successfully applied for fast and easy examination of cell culture supernatants and the investigation of antibody/antigen interactions including kinetics and fluorescence properties. These methods simplify the selection of the optimal antibody. As a target analyte, carbamazepine was chosen. This is a widely used antiepileptic drug which also frequently occurs in the environment. The new antibody enables CBZ determination in the concentration range 0.66–110 µg L-1 within 10 min using a high-throughput microtiter plate-based FPIA, and between 1.4 and 79 µg L-1 within 5 min applying an automated cuvette-based FPIA instrument, and from 0.05–36 µg L-1 using ELISA. The measurements were performed at a non-equilibrium state which improved the sensitivity and selectivity of the assays. Due to low cross-reactivity especially towards the main CBZ metabolite and other pharmaceuticals (<1%), this antibody gives the opportunity for application in medical and environmental analyses.
Suitable labels are at the core of luminescence and fluorescence imaging and sensing. One of the most exciting, yet also controversial, advances in label technology is the emerging development of quantum dots (QDs)inorganic nanocrystals with unique optical and chemical properties but complicated surface chemistryas in vitro and in vivo fluorophores. Here we compare and evaluate the differences in physicochemical properties of common fluorescent labels, focusing on traditional organic dyes and QDs. Our aim is to provide a better understanding of the advantages and limitations of both classes of chromophores, to facilitate label choice and to address future challenges in the rational design and manipulation of QD labels.
Enzyme-activatable optical probes are important for future advances in cancer imaging, but may easily suffer from low signal-to-background ratios unless not optimized. To address this shortcoming, numerous mechanisms to modulate the fluorescence signal have been explored.
We report herein newly synthesized probes based on selfimmolative linkers containing chiral J-aggregate-forming dyes.
Signal modulation by formation of chiral J-aggregates is yet unexplored in optical enzyme probe design. The comprehensive characterization of the probes by absorption, CD, fluorescence, and time-resolved fluorescence spectroscopy revealed dye−dye interactions not observed for the free dyes in solution as well as dye−protein interactions with the enzyme. This suggested
that J-aggregate formation is challenging to achieve with current probe design and that interactions of the dyes with the Enzyme may interfere with achieving high signal-to-background ratios. The detailed understanding of the interactions provided herein provides valuable guidelines for the future design of similar probes.
Oligonucleotide-templated reactions (OTRs) between two reactive hybridization probes allow for the detection of a DNA or RNA of interest by exploiting the target molecule as a catalyst of chemical reactions.
The product of such a reaction commonly exhibits distinct fluorescence properties and can be detected by the means of fluorescence spectroscopy. The vast majority of OTR systems utilize organic dyes as fluorescent reporters. However, the use of brighter emitters, such as semiconductor quantum dots (QDs), has potential to improve the sensitivity of detection by providing brighter signals and permitting the use of probes at very low concentrations. Here we report an RNA-templated reaction between two fluorescently labeled peptide nucleic acid (PNA)-based probes, which proceeds on the surface of a QD. The QD-Bound PNA probe bears a cysteine functionality, while the other PNA is functionalized with an organic dye as a thioester. OTR between these probes proceeds through a transfer of the organic dye to the QD and can be conveniently monitored via fluorescence resonance energy transfer (FRET) from the QD to the Cy5. The reaction was performed in a conventional fluorescence microplate reader and permits the detection of RNA in the picomolar range.