Advances in optical single-molecule detection: On the road to super-sensitive bioaffinity assays

The ability to detect low concentrations of analytes and in particular low‐abundance biomarkers is of fundamental importance, e.g., for early‐stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single‐molecule bioaffinity assays. While many review articles have highlighted the potentials of single‐molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real‐world applications as one should expect. This Review provides a theoretical background on single‐molecule—or better digital—assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single‐molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials

Effect of Particle Size and Surface Chemistry of Photon Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin

Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL−1 in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals

Effect of Particle Size and Surface Chemistry of Photon-Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin

Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL(-1) in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals

Preparation and Characterisation of Highly Stable Iron Oxide Nanoparticles for Magnetic Resonance Imaging

Magnetic nanoparticles produced using aqueous coprecipitation usually exhibit wide particle size distribution. Synthesis of small and uniform magnetic nanoparticles has been the subject of extensive research over recent years. Sufficiently small superparamagnetic iron oxide nanoparticles easily permeate tissues and may enhance the contrast in magnetic resonance imaging. Furthermore, their unique small size also allows them to migrate into cells and other body compartments. To better control their synthesis, a chemical coprecipitation protocol was carefully optimised regarding the influence of the injection rate of base and incubation times. The citrate-stabilised particles were produced with a narrow average size range below 2nm and excellent stability. The stability of nanoparticles was monitored by long-term measurement of zeta potentials and relaxivity. Biocompatibility was tested on the Caki-2 cells with good tolerance. The application of nanoparticles for magnetic resonance imaging (MRI) was then evaluated. The relaxivities and ratio calculated from MR images of prepared phantoms indicate the nanoparticles as a promising -contrast probe

A Primer on Luminescence Sensing

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Rapid Detection of Microorganisms Based on Active and Passive Modes of QCM

Label-free immunosensors are well suited for detection of microorganisms because of their fast response and reasonable sensitivity comparable to infection doses of common pathogens. Active (lever oscillator and frequency counter) and passive (impedance analyzer) modes of quartz crystal microbalance (QCM) were used and compared for rapid detection of three strains of E. coli. Different approaches for antibody immobilization were compared, the immobilization of reduced antibody using Sulfo‑SMCC was most effective achieving the limit of detection (LOD) 8 × 104 CFU·mL−1 in 10 min. For the passive mode, software evaluating impedance characteristics in real-time was developed and used. Almost the same results were achieved using both active and passive modes confirming that the sensor properties are not limited by the frequency evaluation method but mainly by affinity of the antibody. Furthermore, reference measurements were done using surface plasmon resonance. Effect of condition of cells on signal was observed showing that cells ruptured by ultrasonication provided slightly higher signal changes than intact microbes

Polymer pencil leads as a porous nanocomposite graphite material for electrochemical applications: The impact of chemical and thermal treatments

Pencil graphite electrodes are a simple, disposable, and low-cost alternative to screen-printed graphite electrodes. In terms of stability and sensitivity, pencil electrodes often outperform conventional carbon ones. This paper discusses and emphasizes the superior properties of polymer pencil graphite electrodes (pPeGEs), which can be exploited in the electrochemical analysis of molecules, such as chlorides, whose signals are missing on common graphite electrodes. The chemical and structural behaviour of pencil leads after exposure to acids (HF, HNO3, HClO4) or organic solvents (CH3CN, CH3Cl) was monitored via X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The electrochemical activity of pristine and treated pPeGEs was studied by the cyclic voltammetry (CV) responses of reversible redox probes [Fe(CN)6]3/4- and [Ru(NH3)6]3+/ 2+. XPS proved the presence of siloxanes in the surface matrix of the pencil leads; this finding relates to the hydrophobic surface character of the electrodes. SEM then provided images of the pencil surfaces with microplates and flakes and revealed the removal of siloxanes upon chemical treatment. The CVs of non-dried and dried pPeGEs displayed surface changes in the polymer matrix, accompanied by water loss. Our study shows that the pPeGE retains the character of a stable graphite sensor when exposed to acids and organic solvents, except for HF and chloroform. The discovered effects explain the electrochemical processes occurring on pPeGEs and can contribute to their application in electrochemical sensing and energy storage

Single Molecule Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive Detection of Diagnostic Biomarkers

The ability to detect disease markers at the single molecule level promises the ultimate sensitivity in clinical diagnosis. Fluorescence-based single-molecule analysis, however, is limited by matrix interference and can only probe a very small detection volume, which is typically not suitable for real world analytical applications. We have developed a microtiter plate immunoassay for counting single molecules of the cancer marker prostate specific antigen (PSA) using photon-upconversion nanoparticles (UCNPs) as labels that can be detected without background fluorescence. Individual sandwich immunocomplexes consisting of (1) an anti-PSA antibody immobilized to the surface of a microtiter well, (2) PSA, and (3) an anti-PSA antibody-UCNP conjugate were counted under a wide-field epifluorescence microscope equipped with a 980 nm laser excitation source. The single-molecule (digital) upconversion-linked immunosorbent assay (ULISA) reaches a limit of detection of 1.2 pg mL(-1) (42 fM) PSA in 25% blood serum, which is about ten times more sensitive than commercial ELISAs, and covers a dynamic range of three orders of magnitude. This upconversion detection mode has the potential to pave the way for a new generation of digital immunoassays

Advances in Optical Single‐Molecule Detection: En Route to Supersensitive Bioaffinity Assays

The ability to detect low concentrations of analytes and in particular low-abundance biomarkers is of fundamental importance, e.g., for early-stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single-molecule bioaffinity assays. While many review articles have highlighted the potentials of single-molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real-world applications as one should expect. This Review provides a theoretical background on single-molecule-or better digital-assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single-molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials

Advances in Optical Single‐Molecule Detection: En Route to Supersensitive Bioaffinity Assays

The ability to detect low concentrations of analytes and in particular low-abundance biomarkers is of fundamental importance, e.g., for early-stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single-molecule bioaffinity assays. While many review articles have highlighted the potentials of single-molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real-world applications as one should expect. This Review provides a theoretical background on single-molecule-or better digital-assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single-molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials