Liquid biopsies are becoming an increasingly important potential replacement for existing biopsy procedures which can be invasive, painful and compromised by tumour heterogeneity. This paper reports a simple electrochemical approach tailored towards point of care cancer detection and treatment monitoring from biofluids using a label free detection strategy. The mutations under test were the KRAS G12D and KRAS G13D mutations which are both important in the development and progression of many human cancers and whose presence correlates with poor outcomes. These common circulating tumour markers were investigated in clinical samples and amplified by standard and specialist PCR methodologies for subsequent electrochemical detection.
Following pre-treatment of the sensor to give a clean surface, DNA probes developed specifically for detection of the KRAS G12D and G13D mutations were immobilized onto low cost carbon electrodes using diazonium chemistry and EDC/NHS coupling. Following functionalisation of the sensor it was possible to sensitively and specifically detect mutant KRAS G12D and G13D PCR product from cancer patients against a background of wild type KRAS DNA from the representative sample. Our findings give rise to the basis of a simple and very low cost system for measuring ctDNA biomarkers in patient samples. The current time to result of the system is 4.5 hours with considerable scope for optimisation and already compares favourably to the UK National Health Service biopsy service where patients can wait weeks for their result.
The paper will report the technical developments we have made in the production of clean carbon surfaces for functionlisation, show the assay performance data for KRAS G12D and G13D with a range of PCR systems and demonstrate the potential for measuring response to treatment offered by the system.

Silver nanoclusters (AgNCs) generated on DNA-templates belong to a new class of fluorescent tags showing excellent brightness, photostability as well as biocompatibility. Thus, AgNCs-DNA have been applied in various applications, from the detection of DNA/RNA, environmental monitoring to bioimaging and cancer therapy. In this work, we report fluorescent AgNCs synthesized using two 1,3-diaza-2-oxophenothiazine (tC)-modified oligonucleotides that contain RET-related sequence CCCCGCCCCGCCCCGCCCCA. The communication compares the absorption and emission properties of the obtained systems with silver nanoclusters synthesized on the unmodified oligonucleotide. First, we showed the optimal conditions for AgNCs-DNA synthesis on three DNA templates: (1) RET20 with the sequence 5'-CCC CGC CCC GCC CCG CCC CA-3'; (2) 19tC with the sequence 5'-CCC CGC CCC GCC CCG CCC tCA-3'; (3) 14tC with the sequence 5'-CCC CGC CCC GCC CtCG CCC CA-3'. Next, the silver nanoclusters were characterized by UV/Vis absorption, fluorescence and circular dichroism spectroscopy. Silver nanoclusters RET19tC-AgNCs and RET14tC-AgNCs indicated the several times higher fluorescence intensities in the long-wave emission spectra as compared to RET20-AgNCs. Moreover, silver nanoclusters on tC-modified oligonucleotides showed the higher stability over time. The possibility of using of the silver nanoclusters RET19tC-AgNCs and RET14tC-AgNCs for monitoring pH changes would be also demonstrated.

A microfluidic chip for electrochemical impedance spectroscopy (EIS) is presented as biosensor for the detection of cardiac troponin I (cTnI), which is one of the most specific diagnostic serum biomarkers for myocardial infarction. Impedimetric biosensors enable the detection of a variety of analytes, including small molecules, proteins, and cells. As analyte detection is direct and label-free, they allow fast detection of biomarkers, which is essential in the diagnosis of cardiac infarctions to promote a positive outcome. The EIS chip presented here consists of a microscope glass slide serving as base plate, sputtered electrodes, and a polydimethylsiloxane (PDMS) microchannel. The electrode design mainly consists of a working electrode and a counter electrode made of gold. A silver reference electrode can be included, if required. Protocols for electrode functionalization were developed considering a low initial impedance in addition to analyte-specific binding by corresponding antibodies and reduction of non-specific protein adsorption to prevent false-positive signals. Reagents tested for self-assembled monolayers (SAM) on gold electrodes included hydrocarbons with thiol groups and thiolated oligonucleotides. The optimized coating used thiolated single-strand DNA (ssDNA) and 1,4-benzenedithiol on the working electrode and 1,4-benzenedithiol on the counter electrode. After hybridization with corresponding ssDNA carrying an amino group, the reaction with glutaric anhydride led to carboxyl groups, on which anti-cTnI antibody was covalently coupled using carbodiimide chemistry. The PDMS microchannel was bonded on the glass slide with the functionalized electrodes, and the completed EIS chip was connected to the readout system. Sampling with human serum albumin (HSA), 1000 ng/mL, led to negligible signal changes, while sampling with cTnI, 1 ng/mL, led to a significant signal shift in the Nyquist plot. Sampling and measurement took only a few minutes. The results were promising regarding a future cost-effective biosensor array chip for the rapid detection of clinically relevant biomarkers in real samples.

Currently numerous studies are aimed at developing non-invasive technologies for analyzing blood glucose concentration. This opportunity will allow for the prevention and early diagnosis of the population to identify violations of carbohydrate metabolism, which will lead to a decrease in the amount of diabetes mellitus, as well as save huge costs associated with consumables. However, despite the intensive development of non-invasive blood glucose meters, this technology still does not exist. The complexity of the task is due to the huge number of factors affecting the reading of a non-invasive glucometer, which must be optimized to obtain an effective device. First of all, this is due to the presence of a protective skin and muscle cover of a human. As a rule, the skin and parameters of the internal environment introduce significant errors in the measured data. The authors of the project see an original solution to this problem in the study of the so-called near-field effect. The near field of the emitter penetrates deep enough since it does not experience significant absorption in the conductive medium. This will maximize the signal-to-noise ratio.

The work demonstrates a model of a near-field sensor, which has a high penetration of electromagnetic waves into highly absorbing media at a small sensor size (the diameter is 25 mm, the thickness is 0.76 mm). Based on the experimentally obtained data of the dielectric constant for glucose concentrations of 1.2-10 mmol/l, the reflected signal is shown during mathematical modeling of the sensor in the frequency range 0.5-5 GHz. The influence of other biological tissues (dermis, epidermis, fat layer, muscles) and their influence on the near field of the sensor and the received data are also demonstrated.

Liquid biopsy has emerged as a potential supplement of traditional biopsy for early cancer diagnosis. It consists in monitoring the level of tumour biomarkers present in appropriate bodily fluids. Consequently, the development of non-invasive, easy-to-use and inexpensive methods for the detection of biomarkers is highly demanded for improving diagnosis and minimizing the mortality of cancer. In this context, electrochemical platforms have demonstrated great potential due to their high sensitivity, low cost, fast response as well as compatibility with point-of-care (POC) testing, what contributes to decentralize the analyses and favours the implementation of screening studies. Among them, personal glucose meter (PGM) employed daily in the measurement of blood glucose is the most used analytical method worldwide. Taking advantage of this mature technology, PGM has been recently applied to quantitate a wide range of non-glucose analytes by establishing a relationship between their recognition and glucose generation. This strategy is of particular interest for monitoring tumour biomarkers since PGMs are already approved by international agencies for application to clinical practice, thus paving the way for the launch of the new device to the market.

Thus motivated, we describe the development of a hybridization assay for the detection of prostate cancer antigen-3 or PCA3, a urinary RNA biomarker for prostate cancer diagnosis. Specifically, a sandwich-type genoassay has been implemented onto magnetic microparticles functionalized with streptavidin in combination with a PGM and alkaline phosphatase (ALP) as a glucose-generating enzyme for signal transduction. Moreover, in order to boost the method sensitivity, two fluorescein-tagged reporting probes has been designed for incorporation of two antifluorescein-ALP conjugates per target analyte. This enzyme catalyses the conversion of glucose-1-phosphate into glucose, whose concentration is subsequently determined with a glucometer. The resulting portable approach allows the reliable detection of PCA3 at picomolar levels.