Zdeněk Farka

Nanoparticle-Based Immunochemical Biosensors and Assays: Recent Advances and Challenges

We review the progress achieved during the recent five years in immunochemical biosensors (immunosensors) combined with nanoparticles for enhanced sensitivity. The initial part introduces antibodies as classic recognition elements. The optical sensing part describes fluorescent, luminescent, and surface plasmon resonance systems. Amperometry, voltammetry, and impedance spectroscopy represent electrochemical transducer methods; electrochemiluminescence with photoelectric conversion constitutes a widely utilized combined method. The transducing options function together with suitable nanoparticles: metallic and metal oxides, including magnetic ones, carbon-based nanotubes, graphene variants, luminescent carbon dots, nanocrystals as quantum dots, and photon up-converting particles. These sources merged together provide extreme variability of existing nanoimmunosensing options. Finally, applications in clinical analysis (markers, tumor cells, and pharmaceuticals) and in the detection of pathogenic microorganisms, toxic agents, and pesticides in the environmental field and food products are summarized.

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.

Competitive Upconversion-Linked Immunosorbent Assay for the Sensitive Detection of Diclofenac

Photon-upconverting nanoparticles (UCNPs) emit light of shorter wavelength under near-infrared excitation and thus avoid optical background interference. We have exploited this unique photophysical feature to establish a sensitive competitive immunoassay for the detection of the pharmaceutical micropollutant diclofenac (DCF) in water. The so-called upconversion-linked immunosorbent assay (ULISA) was critically dependent on the design of the upconversion luminescent detection label. Silica-coated UCNPs (50 nm in diameter) exposing carboxyl groups on the surface were conjugated to a secondary anti-IgG antibody. We investigated the structure and monodispersity of the nanoconjugates in detail. Using a highly affine anti-DCF primary antibody, the optimized ULISA reached a detection limit of 0.05 ng DCF per mL. This performance came close to a conventional enzyme-linked immunosorbent assay (ELISA) without the need for an enzyme-mediated signal amplification step. The ULISA was further employed for analyzing drinking and surface water samples. The results were consistent with a conventional ELISA as well as liquid chromatography-mass spectrometry (LC-MS).

Detection of Aerosolized Biological Agents Using the Piezoelectric Immunosensor

Airborne microorganisms are a major cause of respiratory diseases. Detection of pathogenic bacteria in the form of bioaerosols is required not only in peacetime but also in the threat of biological attacks. The label-free and direct detection of aerosolized biological agents is presented here. A desktop bioaerosol chamber for safe work with aerosolized microbial cells was constructed, and its functionality was tested. The model organisms (Escherichia coli) were disseminated using an aerosol generator in the chamber filled with either common laboratory indoor air or sterile air. The particles from the generated aerosol were collected using the cyclone SASS 2300, suspended in buffer and then analyzed using the piezoelectric immunosensor modified with specific capture antibodies. The frequency shifts indicated presence of the model biological agent with limit of detection of 1.45 × 10(4) CFU·L(-1) of air. The total time from sample collection to detection was 16 min. The system was fully automated and controlled remotely through a local network.

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.

Carboxyl-rich plasma polymer surfaces in surface plasmon resonance immunosensing

Stable carboxyl-rich plasma polymers (PPs) were deposited onto the gold surface of surface plasmon resonance (SPR) chips under conditions that were chosen based on lumped kinetic model results. Carboxyl-rich films are of high interest for bio-applications thanks to their high reactivity, allowing the formation of covalent linkages between biomolecules and a surface. Accordingly, the monoclonal antibody, specific to human serum albumin (HSA), was immobilized and the performance of SPR immunosensors was evaluated by the immunoassay flow test. The developed sensors performed high level of stability and provided selective and high response to the HSA antigen solutions. The achieved results confirmed that the presented methodologies for the grafting of biomolecules on the gold surfaces have great potential for biosensing applications.

Short-term assessment of cadmium toxicity and uptake from different types of Cd-based Quantum Dots in the model plant Allium cepa L.

We report on the toxicity and bioaccumulation of three different types of Cd-based quantum dots (QDs), dispersed in aqueous medium, for a model plant Allium cepa L. It is believed that encapsulation of nanoparticles should reduce their toxicity and increase their stability in different environments; in this work we studied how QD encapsulation affects their phytotoxicity. Core, core/shell, and core/shell/shell QDs (CdTe, CdTe/ZnS, and CdTe/CdS/ZnS QDs capped by 2-mercaptopropionic acid) were tested and CdCl2 was used as a positive control. After 24-h and 72-h exposure, total Cd content (MCd) and bioaccumulation factors (BAFs) were determined in all parts of A. cepa plants (roots, bulb, shoot), and the total length of the root system was monitored as a toxicity end-point. Measurements of total Cd content versus free Cd2+ content (with Differential Pulse Voltammetry, DPV) in exposure media showed differences in chemical stability of the three QD types. Correspondingly, selected QDs showed different toxicity for A. cepa and different Cd bioaccumulation patterns. CdTe QDs were the most toxic; their effect was similar to CdCl2 due to the release of free Cd2+, which was confirmed by the DPV measurements. Plants exposed to CdTe QDs also bioaccumulated the most Cd among all QD exposure groups. CdTe/ZnS QDs showed no toxicity and very low bioaccumulation of Cd in A. cepa; the main source of measured Cd in the plants were QDs adsorbed on their roots, which was confirmed by fluorescence microscopy. On the contrary, CdTe/CdS/ZnS QD toxicity and bioaccumulation patterns were similar to those of CdTe QDs and pointed to unstable CdS/ZnS shells.

Time-Dependent Growth of Silica Shells on CdTe Quantum Dots

The purpose of this study is to investigate the time dependent growth of silica shells on CdTe quantum dots to get their optimum thicknesses for practical applications. The core/shell structured silica-coated CdTe quantum dots (CdTe/SiO2 QDs) were synthesized by the Ströber process, which used CdTe QDs co-stabilized by mercaptopropionic acid. The coating procedure used silane primer (3-mercaptopropyltrimethoxysilane) in order to make the quantum dots (QDs) surface vitreophilic. The total size of QDs was dependent on both the time of silica shell growth in the presence of sodium silicate, and on the presence of ethanol during this growth. The size of particles was monitored during the first 72 h using two principally different methods: Dynamic Light Scattering (DLS), and Scanning Electron Microscopy (SEM). The data obtained by both methods were compared and reasons for differences discussed. Without ethanol precipitation, the silica shell thickness grew slowly and increased the nanoparticle total size from approximately 23 nm up to almost 30 nm (DLS data), and up to almost 60 nm (SEM data) in three days. During the same time period but in the presence of ethanol, the size of CdTe/SiO2 QDs increased more significantly: up to 115 nm (DLS data) and up to 83 nm (SEM data). The variances occurring between silica shell thicknesses caused by different methods of silica growth, as well as by different evaluation methods, were discussed.

Physical and electrochemical aspects of bio- and nanomaterials

This Special Issue of Chemical Monthly is dedicated to contributions presented at the 15th Workshop of Physical Chemists and Electrochemists (WOPCE) which took place in Brno, Czech Republic, in May 2015. This two-day conference has been organized since 2000, and the last 3 years of the WOPCE were held in the new campus of Masaryk University. All participants presented the latest research in an inspiring lecture session or in the traditionally highly stimulating poster session. The konference scope changes slightly throughout the years, but physical chemistry together with electrochemistry always remains as the main WOPCE topic. The aim of this meeting is to show students and young researchers that physical chemistry is broadly defined and of fundamental importance for the development of interdisciplinary areas that link synthetic, structural, biological, materials and theoretical chemistry together. As the organizers of this meeting, we want to underline the fact that the development of science contributes to intensified interdisciplinary collaboration, thus closely linking scientific research in the fields of both life and materials science. An integral part of the WOPCE is the Young Researchers’ Section which gives the opportunity not only to present and discuss results, but also to compete for the best and most interesting presentation.