A 96-Well LED Array for Multiplexed Photoelectrochemical Detection of Nucleic Acids.

Photoelectrochemical detection of nucleic acid-based cancer biomarkers offers opportunities for highly sensitive, selective, and fast quantitative detection using low-cost measurement instruments. In order to establish itself as a standard method for identifying and quantifying nucleic acids, we have developed a multiplexing strategy using LED technology for photoelectrochemical detection in 96 samples simultaneously. A dedicated setup based on the 96-well plate configuration with a custom-made 96-well LED array was developed. Subsequently, a proof-of-concept study was performed for three miRNAs that are associated with prostate cancer, i.e., miRNA-141, miRNA-145, and miRNA-375. First, measurements with photosensitizer chlorin e6 and redox reporter hydroquinone free in solution proved the proper functioning of the multiplexed detection. Second, the photoelectrochemical detection of the three miRNAs at 24 nM levels was successfully demonstrated. Thereafter, linear calibration curves (R2 > 0.9 for all analytes) were made with plasma spiked with 8-500 pM miRNA. This work presents the first system for multiplexed high-throughput photoelectrochemical detection, allowing it potentially to become a cost-effective and faster alternative to RT-qPCR and gene sequencing techniques in the future.

Singlet oxygen-based photoelectrochemical detection of miRNAs in prostate cancer patients' plasma: A novel diagnostic tool for liquid biopsy.

Dysregulation of miRNA expression occurs in many cancers, making miRNAs useful in cancer diagnosis and therapeutic guidance. In a clinical context using methods such as polymerase chain reaction (PCR), the limited amount of miRNAs in circulation often limits their quantification. Here, we present a PCR-free and sensitive singlet oxygen (1O2)-based strategy for the detection and quantification of miRNAs in untreated human plasma from patients diagnosed with prostate cancer. A target miRNA is specifically captured by functionalised magnetic beads and a detection oligonucleotide probe in a sandwich-like format. The formed complex is concentrated at the sensor surface via magnetic beads, providing an interface for the photoinduced redox signal amplification. The detection oligonucleotide probe bears a molecular photosensitiser, which produces 1O2 upon illumination, oxidising a redox reporter and creating a redox cycling loop, allowing quantification of pM level miRNA in diluted human plasma within minutes after hybridisation and without target amplification.

Development of an electrochemiluminescent oligonucleotide-based assay for the quantification of prostate cancer associated miR-141-3p in human serum.

MicroRNAs (miRNAs) are small oligonucleotides (18-25 bases), biologically relevant for epigenetic regulation of key processes, particularly in association with cancer. Research effort has therefore been directed towards the monitoring and detection of miRNAs to progress (early) cancer diagnoses. Traditional detection strategies for miRNAs are expensive, with a lengthy time-to-result. In this study we develop an oligonucleotide-based assay using electrochemistry for the specific, selective and sensitive detection of a circulating miRNA (miR-141) associated with prostate cancer. In the assay, the excitation and readout of the signal are independent: an electrochemical stimulation followed by an optical readout. A 'sandwich' approach is incorporated, consisting of a biotinylated capture probe immobilised on streptavidin-functionalised surfaces and a detection probe labelled with digoxigenin. We show that the assay allows the detection of miR-141 in human serum, even in the presence of other miRNAs, with a LOD of 0.25 pM. The developed electrochemiluminescent assay has, therefore, the potential for efficient universal oligonucleotide target detection via the redesign of capture and detection probes.

Singlet oxygen-based photoelectrochemical detection of DNA.

The current work, designed for the photoelectrochemical detection of DNA, evaluates light-responsive DNA probes carrying molecular photosensitizers generating singlet oxygen (1O2). We take advantage of their chromophore's ability to produce 1O2 upon photoexcitation and subsequent photocurrent response. Type I, fluorescent and type II photosensitizers were studied using diode lasers at 406 nm blue, 532 nm green and 659 nm red lasers in the presensce and absence of a redox reporter, hydroquinone (HQ). Only type II photosensitizers (producing 1O2) resulted in a noticeable photocurrent in 1-4 nA range upon illumination, in particular, dissolved DNA probes labeled with chlorin e6 and erythrosine were found to give a well-detectable photocurrent response in the presence of HQ. Whereas, Type I photosensitizers and fluorescent chromophores generate negligible photocurrents (<0.15 nA). The analytical performance of the sensing system was evaluated using a magnetic beads-based DNA assay on disposable electrode platforms, with a focus to enhance the sensitivity and robustness of the technique in detecting complementary DNA targets. Amplified photocurrent responses in the range of 70-100 nA were obtained and detection limits of 17 pM and 10 pM were achieved using magnetic beads-captured chlorin e6 and erythrosine labeled DNA probes respectively. The presented novel photoelectrochemical detection can further be optimized and employed in applications for which enzymatic amplification such as polymerase chain reaction (PCR) is not applicable owing to their limitations and as an effective alternative to colorimetric detection when rapid detection of specific nucleic acid targets is required.

Optimized Photoelectrochemical Detection of Essential Drugs Bearing Phenolic Groups.

The World Health Organization (WHO) model "List of Essential Medicines" includes among indispensable medicines antibacterials and pain and migraine relievers. Monitoring their concentration in the environment, while challenging, is important in the context of antibiotic resistance as well as their production of highly toxic compounds via hydrolysis. Traditional detection methods such as high-performance liquid chromatography (HPLC) or LC combined with tandem mass spectrometry or UV-vis spectroscopy are time-consuming, have a high cost, require skilled operators and are difficult to adapt for field operations. In contrast, (electrochemical) sensors have elicited interest because of their rapid response, high selectivity, and sensitivity as well as potential for on-site detection. Previously, we reported a novel sensor system based on a type II photosensitizer, which combines the advantages of enzymatic sensors (high sensitivity) and photoelectrochemical sensors (easy baseline subtraction). Under red-light illumination, the photosensitizer produces singlet oxygen which oxidizes phenolic compounds present in the sample. The subsequent reduction of the oxidized phenolic compounds at the electrode surface gives rise to a quantifiable photocurrent and leads to the generation of a redox cycle. Herein we report the optimization in terms of pH and applied potential of the photoelectrochemical detection of the hydrolysis product of paracetamol, i.e., 4-aminophenol (4-AP), and two antibacterials, namely, cefadroxil (CFD, ß-lactam antibiotic) and doxycycline (DXC, tetracycline antibiotic). The optimized conditions resulted in a detection limit of 0.2 µmol L-1 for DXC, but in a 10 times higher sensitivity, 20 nmol L-1, for CFD. An even higher sensitivity, 7 nmol L-1, was noted for 4-AP.