A novel ferrocene-derived substrate for the ratiometric electrochemical detection of alkaline phosphatase (ALP) was designed and synthesised. It was demonstrated to be an excellent electrochemical substrate for the ALP-labelled enzyme-linked immunosorbent assay (ELISA).
Diagnostic assays that rely on molecular interactions have come a long way; from initial reversible detection systems towards irreversible reaction indicator-based methods. More recently, the emergence of innovative molecular amplification methodologies has revolutionised sensing, allowing diagnostic assays to achieve ultra-low limits of detection. There have been a significant number of molecular amplification approaches developed over recent years to accommodate the wide variety of analytes that require sensitive detection. To celebrate this achievement, this comprehensive critical review has been compiled to give a broad overview of the many different approaches used to attain amplification in sensing with an aim to inspire the next generation of diagnostic assays looking to achieve the ultimate detection limit. This review has been created with the focus on how each conceptually unique molecular amplification methodology achieves amplification, not just its sensitivity, while highlighting any key processes. Excluded are any references that were not found to contain an obvious molecular amplifier or amplification component, or that did not use an appropriate signal readout that could be incorporated into a sensing application. Additionally, methodologies where amplification is achieved through advances in instrumentation are also excluded. Depending upon the type of approach employed, amplification strategies are divided into four categories: target, label, signal or receptor amplification. More recent, more complex protocols combine a number of approaches and are therefore categorised by which amplification component described within was considered as the biggest advancement. The advantages and disadvantages of each methodology are discussed along with any limits of detection, if stated in the original article. Any subsequent use of the methodology within sensing or any other application is also mentioned to draw attention to its practicality. The importance of amplification within sensing is wholly emphasised while perspectives on the future direction of the field are also shared.
Hydrogen peroxide (HO) detection is of high importance as it is a versatile (bio)marker whose detection can indicate the presence of explosives, enzyme activity and cell signalling pathways. Herein, we demonstrate the rapid and accurate ratiometric electrochemical detection of HO using disposable screen-printed electrodes through a reaction-based indicator assay. Ferrocene derivatives equipped with self-immolative linkers and boronic acid ester moieties were synthesised and tested, and, through a thorough assay optimisation, the optimum probe showed good stability, sensitivity and selectivity towards HO. The optimised conditions were then applied to the indirect detection of glucose via an enzymatic assay, capable of distinguishing 10 μM from the background within minutes.
We report on a novel strategy for DNA aptamer immobilization to develop sensitive electrochemical detection of a protein biomarker, with prostate specific antigen (PSA) as a case biomarker. Thiolated single-stranded DNA (ssDNA) was co-immobilized with 3-mercapto-1-propanol on gold electrodes, and used as a scaffold for DNA aptamer attachment through hybridization of the aptamer overhang (so-called "DNA-directed immobilization aptamer sensors", DDIAS). In the approach, the complementary DNA aptamer against PSA was assembled by the probe ssDNA onto the electrode to detect PSA; or the probe ssDNA directly hybridized with a complementary DNA aptamer/PSA complex following their pre-incubation in solution, so-called 'on-chip' and 'in-solution' methods, respectively. A double stranded DNA intercalator with a ferrocenyl (Fc) redox marker was synthesized to evaluate the feasibility of the strategy. The results demonstrate that the 'in-solution' method offers a favourable medium (in a homogeneous solution) for the binding between the aptamer and PSA, which shows to be more efficient than the 'on-chip' approach. DDIAS shows promising analytical performance under optimized conditions, with a limit of detection in the range of fM and low non-specific adsorption.
A new label-free electrochemical DNA (E-DNA) biosensor using a custom synthesized ferrocenyl (Fc) double-stranded DNA intercalator as a redox marker is presented. Single-stranded DNA (ssDNA) was co-immobilized on gold electrodes with 6-mecarpto-hexanol to control the surface density of the ssDNA probe, and hybridized with complementary DNA. The binding of the Fc intercalator to dsDNA was measured by differential pulse voltammetry. This new biosensor was optimized to allow the detection of single base pair mismatched sequences, able to detect as low as 10 pM target ssDNA with a dynamic range from 10 pM to 100 nM. DNA extracted from wastewater was analyzed by quantitative polymerase chain reaction targeting human-specific mitochondrial DNA (mtDNA). The aim of this approach is to enable the analysis of population biomarkers in wastewater for the evaluation of public health using wastewater-based epidemiology (WBE). The E-DNA biosensor was employed to detect human-specific mtDNA from wastewater before and after PCR amplification. The results demonstrate the feasibility of detecting human DNA biomarkers in wastewater using the developed biosensor, which may allow the further development of DNA population biomarkers for public health using WBE.
An enzyme-triggered catalytic signal amplification cascade is described through the design of a novel enzyme substrate that selectively activates an organometallic transfer hydrogenation catalyst once triggered.
Rational tuning of ferrocene redox potential is achieved by modulation of the hydrophobicity and correlated to c log P.
Electrochemical biosensors are an increasingly attractive option for the development of a novel analyte detection method, especially when integration within a point-of-use device is the overall objective. In this context, accuracy and sensitivity are not compromised when working with opaque samples as the electrical readout signal can be directly read by a device without the need for any signal transduction. However, electrochemical detection can be susceptible to substantial signal drift and increased signal error. This is most apparent when analysing complex mixtures and when using small, single-use, screen-printed electrodes. Over recent years, analytical scientists have taken inspiration from self-referencing ratiometric fluorescence methods to counteract these problems and have begun to develop ratiometric electrochemical protocols to improve sensor accuracy and reliability. This review will provide coverage of key developments in ratiometric electrochemical (bio)sensors, highlighting innovative assay design, and the experiments performed that challenge assay robustness and reliability.
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