Optimisation and Characterisation of Anti-Fouling Ternary SAM Layers for Impedance-Based Aptasensors

An aptasensor with enhanced anti-fouling properties has been developed. As a case study, the aptasensor was designed with specificity for human thrombin. The sensing platform was developed on screen printed electrodes and is composed of a self-assembled monolayer made from a ternary mixture of 15-base thiolated DNA aptamers specific for human thrombin co-immobilised with 1,6-hexanedithiol (HDT) and further passivated with 1-mercapto-6-hexanol (MCH). HDT binds to the surface by two of its thiol groups forming alkyl chain bridges and this architecture protects from non-specific attachment of molecules to the electrode surface. Using Electrochemical Impedance Spectroscopy (EIS), the aptasensor is able to detect human thrombin as variations in charge transfer resistance (Rct) upon protein binding. After exposure to a high concentration of non-specific Bovine Serum Albumin (BSA) solution, no changes in the Rct value were observed, highlighting the bio-fouling resistance of the surface generated. In this paper, we present the optimisation and characterisation of the aptasensor based on the ternary self-assembled monolayer (SAM) layer. We show that anti-fouling properties depend on the type of gold surface used for biosensor construction, which was also confirmed by contact angle measurements. We further studied the ratio between aptamers and HDT, which can determine the specificity and selectivity of the sensing layer. We also report the influence of buffer pH and temperature used for incubation of electrodes with proteins on detection and anti-fouling properties. Finally, the stability of the aptasensor was studied by storage of modified electrodes for up to 28 days in different buffers and atmospheric conditions. Aptasensors based on ternary SAM layers are highly promising for clinical applications for detection of a range of proteins in real biological samples

Sensitive and selective Affimer-functionalised interdigitated electrode-based capacitive biosensor for Her4 protein tumour biomarker detection

A novel Affimer-functionalised interdigitated electrode-based capacitive biosensor platform was developed for detection and estimation of Her4, a protein tumour biomarker, in undiluted serum. An anti-Her4 Affimer with a C-terminal cysteine was used to create the bio-recognition layer via self-assembly on gold interdigitated electrodes for the sensor fabrication. Electrochemical impedance spectroscopy (EIS) in the absence of redox markers was used to evaluate the sensor performance by monitoring the changes in capacitance. The Affimer sensor in buffer and in undiluted serum demonstrated high sensitivity with a broad dynamic range from 1 pM to 100 nM and a limit of detection lower than 1 pM both in buffer and in serum. Furthermore, the Affimer sensor demonstrated excellent specificity with negligible interference from serum proteins, suggesting resilience to non-specific binding. The sensing ability of the present Affimer sensor in spiked undiluted serum suggests its potential for a new range of Affimer-based sensors. The fabricated Affimer sensor can thus be further adapted with other probes having affinities to other biomarkers for a new range of biosensors

Detection of prostate cancer biomarker using molecularly imprinted polymers

Successful treatment of prostate cancer (PCa) depends on early diagnosis and screening, which currently relies on the measurement of serum prostate specific antigen (PSA) levels. The overarching aim of the project was to generate molecularly imprinted polymers for PCa biomarkers, with subsequent integration with a sensing platform to allow for rapid, point of care detection and monitoring. The initial work involved the use of simple PSA epitopes for epitope imprinting using conventional imprinting techniques. A four amino acid sequence from the Cterminus of PSA was imprinted with MAA, Aam and Urea monomers to obtain bulk imprinted polymers. Apparent Kd of 102 μM, 154 μM, 194 μM was obtained for MAA, AAm, Urea based bulk mini-MIPs respectively. Epitope imprinting was further developed using a surface imprinting approach, via electropolymersiation of dopamine to detect an epitopic sequence from pro-PSA. An improvement in Kd from bulk-imprinted polymers, with an apparent Kd of 2.9 μM was obtained with the surface electrochemical MIP sensor. However, both epitope imprinting technique lacked sensitivity to measure clinical relevant concentrations of PSA (nM range). As a consequence, a more sophisticated technique called hybrid imprinting was developed to build an electrochemical MIP sensor. Hybrid MIP imprinting utilised an aptamer with established affinity towards PSA to trap the aptamer-PSA complex into a surface grown electropolymer (polydopamine). The resulting aptamer lined polymer pockets exhibited high selectivity and affinity towards PSA (apparent Kd 0.3 nM). The apta-MIP sensor was also able to discriminate from a homologous protein (human Kallikrein 2) and was resilient to fouling from serum proteins. The apta-MIP sensor was further translated to a MOSFET device whereby successful detection of PSA at clinically relevant concentration was obtained in human plasma. Although good sensitivity and selectivity was obtained with the hybrid-MIP sensors, further research is required to understand the binding mechanism of the template to the MIP

Towards implanted biosensors: methods for miniaturising and protecting peptide-based electrochemical sensors

There is real interest in developing selective and sensitive tools to detect protease activity; these play pivotal roles in cancer progression with changes in their amounts and types linked to several pathological processes such as tumour formation, evolution and even suppression. Peptide-based electrochemical assays have been shown to offer several potential advantages over other tools and techniques for development into sensing systems. However, their implantation and use in vivo is complex as they face serious limitations when considering two vital requirements for implantation: sensor miniaturisation for ready implantation and localised measurement and controlled anti-biofouling protection. This study presents the investigation and analysis of these miniaturisation- and protection-related issues and the development of solutions as key steps towards the localised in vivo application and measurements. The first part of the presented work focuses on the potential for assay miniaturisation. This used commercial platinum microelectrodes which were modified with self-assembled monolayer (SAM)-based protease sensing probes. Building on previous macroelectrode studies, which have explored and optimised the use of different SAM structures, redox labelling, anchor type and various spacers of different lengths, further optimisation was carried out with the aim of developing and defining an optimum microelectrode protocol. Comparison of the quantitative analytical performance of macro- and microelectrode systems established the feasibility of developing miniaturised platforms for efficient and clinically-relevant protease detection. Interestingly, significant differences were observed such as an enhanced reproducibility and decreased cleavage rate for the microelectrodes, which were thought to be indicative of variation in the SAM probe film structure on these electrode surfaces caused by differences in film deposition kinetics. This decreased cleavage (response) rate was mitigated by measurement at normal body temperature which was shown to increase kinetics and suggested the possibility of more rapid in vivo sensing. These miniaturisation findings on commercial microelectrodes were translated to in-house microelectrodes fabricated as platinum thin film-on-silicon chips. Initial results showed reduced SAM probe stability. As the use of stronger SAM probe anchoring (through tripodanchored probes) did not solve this problem, the underlying reason was attributed to structural differences between the surfaces of commercial and in-house electrodes, resulting in enhanced Pt detachment in the latter. Increasing metal film thickness and post-fabrication annealing did not completely overcome this problem, and the remaining decrease in stability was attributed to increased Pt surface roughness and destabilisation through successive electrochemical oxidation and reduction during acidic cleaning. An alternative electrochemical reductive cleaning method was thus developed and tested on enhanced electrode sensing systems; arrays of microelectrodes (MEA) and microcavity nanoband edge electrodes (MNEE) were fabricated, cleaned using this reductive method, characterised using typical redox couples and then tested for protease sensing. Gratifyingly, these systems were found to be sufficiently reproducible and stable for sensing. Although functionalised MNEEs achieved significantly higher current densities, there was no great enhancement of response rate from decreasing electrode size from micro to nano, consistent with the fact that diffusional transport is not the rate determining step in this cleavage reaction. Given the variability of probe film deposition characteristics and the resulting cleavage rates, the applicability of potential-controlled SAM probe deposition for controlling probe film formation was investigated as a proof-of-concept study. The second part of this work concentrated on the development of a sensor protection and activation strategy against biofouling. A pH-triggered dissolvable polymeric coating was dropcast onto clean and probe-modified electrodes and then characterised in terms of the delayed activation time, enhancement in anti-biofouling properties and retention of sensing characteristics. These results demonstrated that reproducible delayed sensor activation was achieved and controlled by optimising parameters such as coating thickness, homogeneity and density through the coating method and temperature. Comparative evaluation of polymercoated and uncoated probe-modified electrodes in a biologically relevant medium also revealed significant improvement in their anti-biofouling characteristics