Industry Viable Electrochemical DNA Detection Sensor Architecture Via a Stem-Loop Methylene Blue Redox Reporter and Rapid in Situ Probe Mobilization Method

Pharmacogenomics is one of the endeavours in the modern acute health care sector due to the complexity of the drug reactions associated with the patients' genetic markers[1]. Therefore, the availability of patient pharmacogenetic marker testing at the point-of-care (POC) setting will help the clinicians to prescribe and personalize the medication to ensure maximum efficiency with minimal adverse drug reactions (ADR). In this regard, we focused on developing a HLA-B*15:02 pharmacogenetic biomarker testing known to cause specific ADRs for Carbamazepine[2]. In this study, disposable screen-printed electrodes (SPE) as the planar sensor surfaces and a sequence-specific stem-loop probe modified with a methylene blue redox (MB) reporter was used to detect the targeted DNA sequence of HLA-B*15:02. The oligo probe with MB reporter at the 3′ end was designed to incorporate 25 nucleotides to detect the specific region. The disulphide-reducing reagent, Tris(2-carboxyethyl) phosphine hydrochloride was introduced to reduce the S-S bond of the oligo probe. The probe immobilization on the working electrode area was performed using the fully automated non-contact dispensing system sciFLEXARRAYER SX by dispensing an array of droplets of 50 µM probe mixture into accurate positioning of the gold working electrode area of SPEs (220AT DropSens). Around 20 SPEs were loaded, and probes were printed during a single batch processing task. Two printing runs were performed, and a total of N=30 biosensors were used in the experimental procedure. The electrochemical behaviour of the sensor was studied by performing the square wave voltammetry (SWV) measurements from -0.4 V to -0.1 V, with a modulation amplitude of 0.02 V and frequency of 25 Hz at a scan rate of 0.026 V/s (Autolab-PGSTAT204). The target hybridization time was fixed at 30 minutes. The p-values ≤ 0.05 was considered statistically significant. The SWV measurements on the sensors with 75 µl, 1X PBS solution (Baseline) showed a well-defined peak at -0.31 V with the standard deviation (SD) of 0.004 V (Ntotal=30) which is consistent with the potential of the MB redox moiety. The statistical t-test has been shown that no statistical significance (p=0.1760) between the two printing runs with the peak heights of 640.5 nA ± 151.2 nA (SD). Upon target hybridization, measured peak heights showed that (a) 152.0 nA ± 23.3 nA for the 50 µM Positive control (oligo with the sequence complementary to the probe), (b) 231.8 nA ± 54.8 nA for the 50 µM Negative control (oligo with the non-complementary sequence to the probe), and (c) 226.2 nA ± 93.2 nA for the Blank-oligo free sensors. This shows ~34% signal suppression in Positive control hybridized sensors, whereas insignificance difference between the Negative and Blank samples hybridized sensors. Furthermore, statistical analysis (one-way ANOVA) show that a statistical significance (p=0.0250) for the Positive vs. Negative, a statistical significance (p=0.0388) for the Positive vs. Blank sample, and no statistical significance (p=0.9793) for the Negative vs. Blank sample. These results confirmed that a complementary target binding event in the stem-loop probe results in a significant signal decrease and the possibility of using this 'signal-off biosensor architecture in detecting HLA-B*15:02 pharmacogenetic biomarker testing platform. It was found that only 6 µl probe mixture was required to cover one sensor. Accordingly, the calculated probe surface density was equivalent to ~1.43×1015 molecules/cm2, which will be 3 order of magnitudes higher than the typical probe surface density (~1012) of biosensors[3]. This high level of probe surface density on the sensors was achieved by printing a high concentration of low volume of redox probes. This will not be possible in the typical wet bench probe immobilization methods. Also, it showed a significantly higher level of faradaic current generated in the biosensor and clear differentiation of the targets. Therefore, this work showcases encouraging results of biosensor architecture, rapid probe immobilization method, the possibility of using the ultra-low volume of probe reagents, and high-throughput productions. This suggested that further development of this pharmacogenetic biomarker testing platform will enhance the technical feasibility and enable the transition of this biosensor from the research to industry. Acknowledgements This work used the Melbourne Centre for Nanofabrication (MCN) in the Victorian Node of the NCRIS-enabled Australian National Fabrication Facility (ANFF). References FDA. Table of Pharmacogenomic Biomarkers in Drug Labeling. Available from: https://www.fda.gov/drugs/science-and-research-drugs/table-pharmacogenomic-biomarkers-drug-labeling. Soraya, G.V., et al., An interdigitated electrode biosensor platform for rapid HLA-B*15:02 genotyping for prevention of drug hypersensitivity. Biosens Bioelectron, 2018. 111: p. 174-183. Ricci, F., et al., Effect of molecular crowding on the response of an electrochemical DNA sensor. Langmuir, 2007. 23(12): p. 6827-6834.